We thank Dr. Shuang Liu for critical reading of the manuscript. The PCL12 construct was a gift form Dr. Christopher D. Link. This work was supported by two National Science Foundation grants (0842708 and 1026958) and an AHA grant (09GRNT2250529) to FS.

1. Construction of Transgenic Worms
<ol>
	<li>Transformation.
	<ol>
		<li>Prepare injection pads. Place a drop of hot, 2% agarose dissolved in water, onto a glass coverslip. Quickly place a second coverslip on the drop and lightly tap it. After the agarose is solidified, slide coverslips apart, and bake the coverslip-pad in a vacuum oven at 80 &deg;C O/N.</li>
		<li>Pull pipettes. We use a Sutter P-97 puller to pull 1/0.5 mm O.D./I.D. borosilicate capillaries with filament. Pipettes are forged with closed tip which is broken open at a later stage.</li>
		<li>Prepare injection mix. Make the injection mixture containing the DNA of interest (20 ng/&mu;l per construct) plus empty plasmid DNA to a final concentration of 150 ng/&mu;l. Centrifuge the injection mixture to remove any contaminant. This step is crucial because contaminants reduce transformation efficiency. Transfer the top 5 &mu;l (debris and other contaminants collect in the bottom) to a fresh tube to be used in injection.</li>
		<li>Load injection mix by capillarity. The bottom of the pipette is immersed in the injection mix. Typically 0.5 &mu;l are sufficient to inject 100 worms.</li>
		<li>Load injection pipette. Insert the pipette onto the holder of the micromanipulator and break it open by rubbing its tip against the edge of a cover slip mounted on a glass slide or alternatively against debris on the agarose pad. This is a crucial step as too large tips damage the worm and too small tips are easily clogged. The quality of the broken tip can be judged by the shape and most importantly by the flow rate. The flow rate can be assessed by the size of the bubbles flowing from the open tip immersed in a drop of halocarbon 700 oil in response to an injection pressure.</li>
		<li>Transfer worms to injection pad. Place a drop of 700 halocarbon oil on an injection pad and transfer several (1 or 2 for beginners) worms to the oil. Use a worm pick to push the worms down onto the pad until they adhere to the agarose. Worms should be oriented in rows with ventral sides facing the same direction. Avoid touching worm's head. If after several attempts the worms fail to adhere to the pad, replace with a fresh pad or increase agarose thickness and/or concentration.</li>
		<li>Insert the pipette into the worm. By moving the stage, position the worm under the pipette. Position the pipette in the central core of the gonad because its cytoplasm is shared by many germ cell nuclei. This increases the likelihood to deliver injected DNA to many progeny. The pipette should lie almost parallel to the worm (~15&deg;-25&deg; angle). Push vertically the tip of the pipette down the worm's body until the skin is depressed. Then gently tap on the manipulator to induce tip's insertion. For best results inject both gonads.</li>
		<li>Inject the DNA solution. Apply pressure to the pipette until the gonad swells. Stop the flow and pull the worm off the pipette. Test the needle for flow and then move to the next worm and repeat injection steps.</li>
		<li>Recover the worms: Add a drop (~10 &mu;l) of recovery buffer on the worms and incubate until the worms begin swimming. Add an equal volume of M9, wait until worms resume swimming and repeat several times until the solution is mostly M9. Individually transfer worms to seeded plates.</li>
	</ol>
	</li>
	<li>Integration.
	<ol>
		<li>Place 40 healthy, well-fed, L4 worms into a fresh plate</li>
		<li>Irradiate with &gamma;-ray with 4,000 rads for 40 min. Transfer irradiated worms (P0) in fresh, OP50 seeded plates (4 worms/plate). Worms can alternatively be irradiated with a dose of 300 J/m2 UVs.</li>
		<li>Transfer 10-20 F1 transformants from each plate to individual plates, label the plates with the corresponding P0 origin.</li>
		<li>Single out 2-4 F2 transformants for each F1 to separate plates. Check the F3 progeny for 100% transmission of the transformation marker. Typically, 1-3% of progeny from an irradiated worm will have an integration event.</li>
		<li>Make stocks of three or more independent transformant lines.</li>
	</ol>
	</li>
</ol>
2. Behavioral Assays
<ol>
	<li>Age-synchronization.
	<ol>
		<li>Grow worms in standard 10 cm NGM plates + OP50 E. coli until a large population of gravid adults is reached (3-5 days).</li>
		<li>Collect the worms in 50 ml Falcon tubes by suspending them in 1 ml M9 buffer.</li>
		<li>Add 5 ml M9 buffer and centrifuge at 450 x g for 3 min. Discard supernatant. Repeat 3-4x.</li>
		<li>At the end of the last centrifugation, remove the supernatant and add 10 volumes of basic hypochlorite solution (0.5 M NaOH, 1% hypochlorite freshly mixed) to the pelleted worms. Incubate at RT for ~10 min. The lysis reaction can be monitored by placing a drop of the lysis reaction on a coverslip and examining the worms under a microscope. When roughly 80% of worms are broken the reaction should be stopped.</li>
		<li>Stop the lysis reaction by adding the same volume of sterile egg buffer.</li>
		<li>Collect the eggs (and carcasses) by centrifugation at 450 x g for 5 min.</li>
		<li>Wash the eggs with sterile egg buffer 2-3x.</li>
		<li>Incubate the eggs O/N in M9 buffer and seed them on standard NGM plates.</li>
	</ol>
	</li>
	<li>Chemotaxis assay.
	<ol>
		<li>Prepare several pieces of agar roughly 0.5x0.5x0.5 cm. We deposit the agar in a 10-cm plate and we cut the agar chunks from there.</li>
		<li>Soak the agar chunk in a solution containing the desired attractant (at saturating, near-saturating concentrations) for 2 hr. Typical attractants are lysine (0.5 M), biotin (0.2 M).</li>
		<li>Deposit an agar chunk in a 10 cm test plate in which the location of a test spot and a control spot have been marked (Figure 1A). Allow equilibration and formation of a gradient O/N (Figure 1B). Prepare 5 plates for a single experiment.</li>
		<li>Prior the experiment add 10 &mu;l of 20 mM NaN3 (anesthetic) to each spot.</li>
		<li>Place 20 age-synchronized worms in the center of the plate. Place the plate in the incubator at 20 &deg;C.</li>
		<li>After 1 hr, count the animals on the test/control spots and calculate the chemotaxis index, (C.I.) as follow: <img alt="Equation 1" fo:content-width=".863in" fo:src="/files/ftp_upload/50435/50435eq1highres.jpg" src="/files/ftp_upload/50435/50435eq1.jpg" /> where N, Ntest and NCnt., indicate the total number of animals, the number of animals in the test spot and the number of animals in the control spot.</li>
	</ol>
	</li>
</ol>
3. Primary Embryonic Cell Culture
<ol>
	<li>Lyse worms as described in age-synchronization.</li>
	<li>Stop the lysis reaction by adding the same volume of sterile egg buffer and centrifuge at 450 x g for 5 min. Gently discard supernatant being careful to not lose pelleted eggs. Repeat 2-3x or until supernatant is clear.</li>
	<li>Resuspend pelleted eggs (and carcasses) in 2 ml sterile egg buffer and add 2 ml of sterile 60% sucrose in egg buffer. Mix this solution until eggs are completely resuspended (as they tend to form clumps under centrifugation) by hand or by vortexing.</li>
	<li>Centrifuge at 450 x g for 15 min.</li>
	<li>Carefully transfer supernatant (containing the eggs) to a sterile tube. Discard pellet which contains carcasses and other by-products of lysis.</li>
	<li>Remove residual sucrose by resuspending the eggs in egg buffer and centrifuging at 450 x g for 5 min. Gently collect and discard the supernatant. Repeat 3x.</li>
	<li>Under a laminar hood resuspend pelleted eggs in sterile egg buffer containing 1 U/ml chitinase at RT to digest the eggshells. After 30 min start to monitor the reaction (under an inverted cell culture microscope). Each batch of chitinase has a slightly different activity. Typically digestion is completed in 1 hr.</li>
	<li>When roughly 70-80% eggshells are digested by chitinase add CM-15 (L-15 cell culture medium containing 10% fetal bovine serum, 50 U/ml penicillin, and 50 &mu;g/ml streptomycin). Dissociate cells using a syringe with a 27 gauge. Filter the cell suspension with a 5.0 &mu;m filter to remove intact embryos, clumps of cells and larvae.</li>
	<li>Pellet the dissociated cell suspension by centrifugation at 450 x g for 15 min. Remove the supernatant and resuspend the pellet in CM-15 cell culture medium.</li>
	<li>Plate dissociated cells on glass cover slips previously coated with peanut lectin (0.1 mg/ml) dissolved in water. Note: cells must adhere to the substrate in order to differentiate.</li>
	<li>Cells can be maintained at RT (16-20 &deg;C) in air for more than 2 weeks.</li>
</ol>

<ol>
	<li>Hardy, J., Allsop, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+deposition+as+the+central+event+in+the+aetiology+of+Alzheimer's+disease.">Amyloid deposition as the central event in the aetiology of Alzheimer's disease.</a> Trends in Pharmacological Sciences. 12 (10), 383-388 (1991).</li><li>Kotzbauer, P. T., Trojanowsk, J. Q., Lee, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lewy+body+pathology+in+Alzheimer's+disease.">Lewy body pathology in Alzheimer's disease.</a> Journal of Molecular Neuroscience. 17 (2), 225-232 (2001).</li><li>Greenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inclusion+body+myositis:+review+of+recent+literature.">Inclusion body myositis: review of recent literature.</a> Current Neurology and Neuroscience Reports. 9 (1), 83-89 (2009).</li><li>Revesz, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sporadic+and+familial+cerebral+amyloid+angiopathies.">Sporadic and familial cerebral amyloid angiopathies.</a> Brain Pathology. 12 (3), 343-357 (2002).</li><li>Hartmann, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+sites+of+intracellular+production+for+Alzheimer's+disease+A+beta40/42+amyloid+peptides.">Distinct sites of intracellular production for Alzheimer's disease A beta40/42 amyloid peptides.</a> Nature Medicine. 3 (9), 1016-1020 (1997).</li><li>Ohnishi, S., Takano, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+fibrils+from+the+viewpoint+of+protein+folding.">Amyloid fibrils from the viewpoint of protein folding.</a> Cellular and Molecular Life sciences: CMLS. 61 (5), 511-524 (2004).</li><li>DL, R. i. d. d. l. e., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+II.">C. elegans II.</a> Cold Spring Harbor Monograph. 1, 1222 (1997).</li><li>Cotella, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxic+role+of+k++channel+oxidation+in+Mammalian+brain.">Toxic role of k+ channel oxidation in Mammalian brain.</a> The Journal of Neuroscience: the official journal of the Society for Neuroscience. 32 (12), 4133-4144 (2012).</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> Proceedings of the National Academy of Sciences of the United States of America. 92 (20), 9368-9372 (1995).</li><li>Cai, S. Q., Sesti, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidation+of+a+potassium+channel+causes+progressive+sensory+function+loss+during+aging.">Oxidation of a potassium channel causes progressive sensory function loss during aging.</a> Nature Neuroscience. 12 (5), 611-617 (2009).</li><li>Yu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guanylyl+cyclase+expression+in+specific+sensory+neurons:+a+new+family+of+chemosensory+receptors.">Guanylyl cyclase expression in specific sensory neurons: a new family of chemosensory receptors.</a> Proc. Natl. Acad. Sci. U.S.A. 94, 3384-3387 (1997).</li><li>Bargmann, C. I., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemosensory+neurons+with+overlapping+functions+direct+chemotaxis+to+multiple+chemicals+in+C.+elegans.">Chemosensory neurons with overlapping functions direct chemotaxis to multiple chemicals in C. elegans.</a> Neuron. 7 (5), 729-742 (1991).</li><li>Caserta, T. 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The authors declare that they have no competing financial interests.

Here we describe a combined approach, to study cellular and molecular aspects of amylopathies using C. elegans. The advantages of this approach include: 1) low cost. C.elegans is maintained in normal Petri dish seeded with bacteria, at room temperature. 2) Powerful genetics. Transgenic animals can be obtained in few months and a wide array of promoter sequences is available to drive expression of the desired gene in specific neurons. 3) Simple, well-characterized, nervous system. C. elegans pos...

Amyloid beta (A&beta;) is a peptide of 36-43 amino acids that is formed after sequential cleavage of the amyloid precursor protein (APP) by &beta; and &gamma; secretases 1. The &gamma; secretase processes the C-terminal end of the A&beta; peptide and is responsible for its variable lengths 5. The most common forms of A&beta; are A&beta;40 and A&beta;42, the latter being more commonly associated to pathologic conditions such as AD 5. At high concentrations A&beta; form &beta;-sheets that aggregate to form amyloid fibrils 6. Fibrils deposits are the main component of senile plaques surrounding neurons. Both plaques and diffusible, non-plaque A&beta; oligomers, are thought to constitute the underlying pathogenic forms of A&beta;.
Laboratory study of neuronal amylopathies is complicated by the fact that these conditions progress with time. Therefore, it is important to develop genetically tractable animal models-complementary to mice-with short life span. These models can be used to elucidate specific aspects of amylopathies-typically cellular and molecular-and by virtue of their simplicity, help to capture the essence of the problem. The worm Caenorhabditis elegans falls is this category. It has a short life span, ~20 days and in addition basic cellular processes including regulation of gene expression, protein trafficking, neuronal connectivity, synaptogenesis, cell signaling, and death are similar to mammalian 7. Unique features of the worm include powerful genetics and lack of a vessel system, which enables to study neuronal damage independently of vascular damage. On the other hand, the lack of a brain limits the use of C. elegans to studying many aspects of neurodegeneration. In addition, the reproduction and identification of anatomical distributions of lesions cannot be performed in this organism. Other limitations include the difficulty to assess both differences in gene expression profiles and impairment of complex behavior and memory function. Here we describe methods to generate C. elegans models of amylopathies.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>1. NGM</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td>3 g</td>
		</tr>
		<tr>
			<td>Bacteriological agar</td>
			<td>AMRESCO</td>
			<td>J637</td>
			<td>17 g</td>
		</tr>
		<tr>
			<td>Bacto-peptone</td>
			<td>AMRESCO</td>
			<td>J636</td>
			<td>2.5 g</td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Bring to 975 ml</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Sterilized by autoclaving, then add the following items and mix well</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M2643</td>
			<td>1 ml of 1 M stock</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C5670</td>
			<td>1 ml of 1 M stock</td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sigma-Aldrich</td>
			<td>C3045</td>
			<td>1 ml of 5 mg/ml stock( in ethanol)</td>
		</tr>
		<tr>
			<td>Potassium phosphate buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>25 ml of 1M stock</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>2. Potassium phosphate buffer</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td>108.3 g</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>P3786</td>
			<td>35.6 g</td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Bring to 1 L</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Sterilized by autoclaving</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>3. M9 buffer</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td>3 g</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>S5136</td>
			<td>6 g</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td>5 g</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M2643</td>
			<td>1 ml of 1 M stock</td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Bring to 1 L</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Sterilized by autoclaving</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>4. Egg buffer (pH 7.3, 340 mOsm)</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td>118 mM</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P5405</td>
			<td>48 mM</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C5670</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M4880</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher Scientific</td>
			<td>BP310</td>
			<td>25 mM</td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Bring to 1 L</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Sterilized by autoclaving</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>5. CM-15</td>
		</tr>
		<tr>
			<td>L-15 culture medium</td>
			<td>Gibco</td>
			<td>11415</td>
			<td>450 ml</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10437-028</td>
			<td>50 ml</td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>Gibco</td>
			<td>15140</td>
			<td>50 units/ml</td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Gibco</td>
			<td>15140</td>
			<td>50 g/ml</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Adjust to 340 mOsm with sucrose then sterile filter into autoclaved bottles and store at 4 &deg;C</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>6. Other Reagents</td>
		</tr>
		<tr>
			<td>Halocarbon 700 oil</td>
			<td>Halocarbon Products</td>
			<td>9002-83-9</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5 &mu;m Acrodisc Syringe Filter</td>
			<td>PALL Co.</td>
			<td>4199</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chitinase</td>
			<td>Sigma-Aldrich</td>
			<td>C6137-5UN</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lectin (peanut)</td>
			<td>Sigma-Aldrich</td>
			<td>L0881-10MG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>S320</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>L5501</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Sigma-Aldrich</td>
			<td>B4639</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium hypochlorite solution</td>
			<td>Sigma-Aldrich</td>
			<td>425044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>71289</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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.

With our protocols we study the effects of human A&beta;42 oligomer on neuronal function 8. A fragment encoding human A&beta;42 and the artificial signal peptide coding sequence of Fire vector pPD50.52 was amplified from construct PCL12 9 using primers that introduced a Sma 1 restriction endonuclease site at the ends. The fragment was then inserted into a construct containing a 2,481-bp flp-6 promoter sequence in the pPD95.75 Fire vector between the unique Sma 1 site <...

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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.

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 ...

a-caenorhabditis-elegans-model-system-for-amylopathy-study

Research

JoVE Journal

Neuroscience

10.4K vistas.  University of Medicine and Dentistry of New Jersey. Se describen métodos para estudiar los aspectos de la amylopathies en el gusano C. elegans. Mostramos cómo construir gusanos expresan Aß humano 42 En las neuronas y la forma de poner a prueba su función en ensayos de comportamiento. Además, muestran cómo obtener cultivos neuronales primarios que pueden ser utilizados para las pruebas farmacológicas.

Video: A Caenorhabditis elegans Model System for Amylopathy Study

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.

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.

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

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. ...

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their locomotory patterns upon detection of diverse environmental stimuli and can modulate their suite of behavioral responses in response to starvation conditions. Nematodes typically exhibit a decreased aversive response when removed from a food source for over 30 min. Observation of behavioral changes in response to a changing nutritional status can provide insight into the mechanisms that regulate the transition from a well-fed to starved state.
We have developed an assay that measures a nematode&#39;s ability to cross an aversive barrier (i.e. copper) then reach a food source over a prolonged period of time. This protocol builds upon previous work by integrating multiple variables in a manner that allows for continued data collection as the organisms shift towards an increasingly starved condition. Moreover, this assay permits an increased sample size so that larger populations of nematodes can be simultaneously evaluated.
Organisms defective for the ability to detect or respond to copper immediately cross the chemical barrier, while wild type nematodes are initially repelled. As wild type worms are increasingly starved, they begin to cross the barrier and reach the food source. We designed this assay to evaluate a mutant that is incapable of responding to diverse environmental cues, including food sensation or detection of aversive chemicals. When evaluated via this protocol, the defective organisms immediately crossed the barrier, but were also incapable of detecting a food source. Hence, these mutants repeatedly cross the chemical barrier despite temporarily reaching a food source. This assay can straightforwardly test populations of worms to evaluate potential pathway defects related to aversion and starvation.

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Here, we present a Caenorhabditis elegans-specific assay designed to evaluate changes in copper aversion behavior and the ability to locate a common food source, as the organism progresses from a well-fed to starved nutritional state.

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic spines have been described for DD GABAergic neurons as the input sites from presynaptic cholinergic neurons in the motor circuit of Caenorhabditis elegans. This synaptic circuit can now serve as a powerful new in vivo model of spine morphogenesis and function that exploits the facile genetics and ready accessibility of C. elegans to live-cell imaging. 
This protocol describes experimental strategies for assessing DD spine structure and function. In this approach, a super-resolution imaging strategy is used to visualize the intricate shapes of actin-rich dendritic spines. To evaluate the DD spine function, the light-activated opsin, Chrimson, stimulates the presynaptic cholinergic neurons, and the calcium indicator, GCaMP, reports the evoked calcium transients in postsynaptic DD spines. Together, these methods comprise powerful approaches for identifying genetic determinants of dendritic spines in C. elegans that could also direct spine morphogenesis and function in the brain.

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are important cellular features of the nervous system. Here live imaging methods are described for assessing the structure and function of dendritic spines in C. elegans.&#160;These approaches support the development of mutant screens for genes that define dendritic spine shape or function.

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic ...

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed orientation. Here we describe methods to label, image, and analyze the microtubule dynamics and growth during the development and regeneration of touch neurons in C. elegans. Using genetically encoded fluorescent reporters of microtubule tips, we imaged the axonal microtubules. The local changes in microtubule behavior that initiates axon regeneration following axotomy can be quantified using this protocol. This assay is adaptable to other neurons and genetic backgrounds to investigate the regulation of microtubule dynamics in various cellular processes.

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

A protocol for imaging the dynamic microtubules in vivo using fluorescently labeled End binding protein has been presented. We described the methods to label, image, and analyze the dynamic microtubules in the Posterior lateral microtubule (PLM) neuron of C. elegans.

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed ...