Name: isolation of specific neuron populations from roundworm caenorhabditis elegans
Uploaded: 2019-08-06T15:00:00
Description: Watch this Scientific Journal Video about Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans at JoVE.com

We thank Dr. Jennifer Fox and the members of the Khalimonchuk laboratory for insightful comments. We acknowledge the support from the National Institutes of Health (R01 GM108975 to O.K. and T32 GM107001-01A1 to E.M.G.).

1. Preparation and collection of aged worms for cell isolation
NOTE: Explained below is the isolation of cholinergic neurons from the transgenic unc-17::GFP strain (OH13083) obtained from the Caenorhabditis Genetics Center (CGC) strain repository at the University of Minnesota. It is imperative to maintain sterile conditions to prevent contamination from fungi or bacteria.
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	<li>Prepare and synchronize worms via the bleaching method as described by T. Stiernagle<sup cla...

     

<ol>
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M., Petrova, K., Benedetti, C., Yang, Y., Ron, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ClpP+mediates+activation+of+a+mitochondrial+unfolded+protein+response+in+C.+elegans.">ClpP mediates activation of a mitochondrial unfolded protein response in C. elegans.</a> Developmental Cell. 13, 467-480 (2007).</li><li>Walter, P., Ron, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+unfolded+protein+response:+from+stress+pathway+to+homeostatic+regulation.">The unfolded protein response: from stress pathway to homeostatic regulation.</a> Science. 334, 1081-1086 (2011).</li><li>Chen, C., Chen, Y., Jiang, H., Chen, C., Pan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuron+aging:+learning+from+C.+elegans.">Neuron aging: learning from C. elegans.</a> Journal of Molecular Signal. 8 (14), 1-10 (2013).</li><li>Germany, E. 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The roundworm C. elegans is a well-established and powerful model to study neuronal health and disease2. With ample genetic tools to manipulate these animals and manageable quantity of precisely mapped various neuron types, a great deal of data can be collected with a relatively small quantity of material. Here, we outline an optimized method to isolate distinct neurons from whole animals. By disrupting the worm&#8217;s outer cuticle proteins, a slurry of various cell types can be isolate...

Over the past several decades, the metazoan model organism Caenorhabditis elegans has been a tremendous asset in the study of neurons, neuronal circuitry and its role in physiological and behavioral responses, and aging-associated neurodegenerative diseases. A unique feature of C. elegans is that the animals are transparent, allowing for the lineage of all adult somatic cells to be mapped1. C. elegans also harbors manageable quantity of neurons, leading to the morphology and connectivity of the nervous system being well understood2. The invariant cell lineage, short lifespan, and abundance of high-throughput genetic tools available for C. elegans make it an ideal model organism for the study of aging within different neuronal populations.
Aging of neurons and neurodegenerative disorders are complex, and each disorder has unique pathological signatures associated with it. However, for many of these disorders, such as Parkinson&#8217;s and Alzheimer&#8217;s disease, a common feature is the progressive load of misfolded proteins3,4,5. In both of these diseases, proteins misfold and aggregate within the cell to cause toxicity, ultimately leading to cell death4,5. For proper aging of neurons, the homeostasis of mitochondria, more specifically protein homeostasis, is important, as perturbations and dyshomeostasis can lead to neurodegeneration6,7,8. Cells are equipped with various mechanisms to maintain protein homeostasis, one being the unfolded protein response (UPR)&#8213;a classical pathway where signals from endoplasmic reticulum (ER) activate intracellular signal cascades, leading to a transcriptional response9. Akin to this UPRER, metazoans display a similar response to loss of mitochondrial protein homeostasis, the so-called mitochondrial UPR (UPRmt)7,8. C. elegans appears to be the most basal organism to have a confirmed and well-defined UPRmt pathway7.
The function/dysfunction of specific neuronal populations within a larger network can be difficult to assess due to their intrinsic connectivity and complexity3. However, there is often a need to study distinct neuronal populations due to cell type-specific diseases with complex pathologies, such as those associated with impaired cellular protein homeostasis. In C. elegans, specific neuronal populations can be genetically manipulated allowing for facile observation in vivo. The nervous system of C. elegans primarily consists of neurons, with a small percentage of glial cells. In adult hermaphrodite worms, there are approximately 302 neurons, subdivided into over 100 different classes2,10. Neurons such as cholinergic neurons predominate in neuromuscular junctions, while dopaminergic neurons are primarily involved in sensation10,11. As both motor activity and sensory abilities decline with age, it is important to detail mechanistic insights about defects in these individual neurons11,12. As such, there is a need for a simple and robust method to isolate intact cells of interest for subsequent ex vivo studies.
Here, we describe an optimized effective method for the rapid isolation of specific neuronal cells expressing green fluorescent protein (GFP) from C. elegans. This isolation method can be performed on larval, juvenile or adult worms. Isolation of cells from larval worms was previously published by Zhang et al.13 and will not be discussed here. An important note here is that all worms need to be at the same life stage to prevent over-digestion of the animal or contamination from animals in different life stages. Through both enzymatic and mechanical disruption of the nematode cuticle, an exoskeleton high in collagen and other structural proteins, a wide variety of cells can be isolated13,14. Cells can then be isolated via flow cytometry or antibody labeled magnetic beads. Typically, RNA is isolated via a phenol and guanidine isothiocyanate method to ensure the enrichment of desired cell population15. With much care these isolated cells can be maintained in a culture flask or multi-well dish. This method represents a unique and powerful tool in the study of specific neurons and has the capacity to isolate live and functional cells for further culturing.

<table>
	<tbody>
		<tr>
			<td>6-well plate</td>
			<td>Fisher Scientific</td>
			<td>12-556-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar, Molecular Biology Grade</td>
			<td>VWR</td>
			<td>A0930</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C5670</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma</td>
			<td>496189</td>
			<td></td>
		</tr>
		<tr>
			<td>Contess Automated Cell Counter</td>
			<td>Invitrogen</td>
			<td>Z359629</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>USBiological</td>
			<td>D8070</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Labs</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Omega Scientific</td>
			<td>FB-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorodeoxyuridine</td>
			<td>Sigma</td>
			<td>F0503</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>VWR</td>
			<td>BDH1133</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Amresco</td>
			<td>O395</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>USBiological</td>
			<td>P5110</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark Professionals</td>
			<td>7552</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium</td>
			<td>Gibco</td>
			<td>21083027</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>USBiological</td>
			<td>M2090</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4&#183;7H2O</td>
			<td>USBiological</td>
			<td>S5199</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>VWR</td>
			<td>X190</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOCl (Bleach)</td>
			<td>Clorox</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Amresco</td>
			<td>O583</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomicen</td>
			<td>Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone Y</td>
			<td>USBiological</td>
			<td>P3306</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase E</td>
			<td>Sigma</td>
			<td>7433</td>
			<td>protease mixture from Streptomyces griseus</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Amresco</td>
			<td>O227</td>
			<td></td>
		</tr>
		<tr>
			<td>SMT1-FLQC fluorescence stereomicroscope</td>
			<td>Tritech Research</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>USBiological</td>
			<td>S8010</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript IV One-Step synthesis kit</td>
			<td>ThermoFisher</td>
			<td>12594025</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol</td>
			<td>Invitrogen</td>
			<td>15596026</td>
			<td>phenol and guanidine isothiocyanate solution</td>
		</tr>
		<tr>
			<td>Trypan Blue Stain</td>
			<td>Invitrogen</td>
			<td>T10Z82</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-GFP magnetic beads</td>
			<td>MBL</td>
			<td>D153-11</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of specific neuron populations from roundworm caenorhabditis elegans

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological conditions. Post-mitotic neurons represent a major cell type affected by aging. Although multiple mammalian models of neuronal aging exist, they are challenging and expensive to establish. The roundworm Caenorhabditis elegans is a powerful model to study neuronal aging, as these animals have short lifespan, an available robust genetic toolbox, and well-cataloged nervous system. The method presented herein allows for seamless isolation of specific cells based on the expression of a transgenic green fluorescent protein (GFP). Transgenic animal lines expressing GFP under distinct, cell type-specific promoters are digested to remove the outer cuticle and gently mechanically disrupted to produce slurry containing various cell types. The cells of interest are then separated from non-target cells through fluorescence-activated cell sorting, or by anti-GFP-coupled magnetic beads. The isolated cells can then be cultured for a limited time or immediately used for cell-specific ex vivo analyses such as transcriptional analysis by real time quantitative PCR. Thus, this protocol allows for rapid and robust analysis of cell-specific responses within different neuronal populations in C. elegans.

The protocol described here allows for specific isolation of unc-17::GFP-positive cholinergic neurons from the roundworm C. elegans for subsequent ex vivo studies such as cell type-specific gene expression profiling and eventual short-term culturing for patch-clamp electrophysiology measurements.
Figure 1 shows unc-17::GFP-positive cholinergic neurons in their normal setti...

Watch this Scientific Journal Video about Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans at JoVE.com

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

Here, we present a protocol for simple isolation of specific groups of live neuronal cells expressing green fluorescent protein from transgenic Caenorhabditis elegans lines. This method enables a variety of ex vivo studies focused on specific neurons and has the capacity to isolate cells for further short-term culturing.

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological ...

This protocol allows for the seamless isolation, culturing, and interrogation of transcriptional responses in a specific cell lineage from C.elegans. The main advantage of this protocol is the high level of adaptability to different life stages of worms or specific cell types to be isolated. Demonstrating the procedure will be myself and Nataliya Zahayko, a technician from our laboratory.After collecting the animals, centrifuge them at 1600 times g for five minutes. Remove all of the supernatant and resuspend the worms in one milliliter of M9 media. Transfer the suspension to a 1.5-milliliter microcentrifuge tube.Centrifuge at 1600 times g for five minutes to pellet the worms. Then add 200 microliters of SDS-DTT buffer and incubate at room temperature for five minutes. The worms should now appear to be wrinkled along the body if viewed under a light microscope.Next, add 800 microliters of ice-cold isolation buffer and mix by gently flicking the tube. Centrifuge at 13, 000 times g and at four degrees Celsius for one minute to pellet the worms. Remove the supernatant and wash with one milliliter of isolation buffer.Repeat the centrifugation and wash process a total of five times, making sure to carefully remove the isolation buffer each time. After this, add 100 microliters of protease mixture from Streptomyces griseus, dissolved in isolation buffer, to the pellet. Incubate at room temperature for 10 to 15 minutes, making sure to apply mechanical disruption by pipetting up and down 60 to 70 times with a 200-microliter micropipette tip against the bottom of the tube.To determine the stage of digestion, remove one to five microliters of the digestion mixture, drop it onto a glass slide, and inspect it using a tissue culture microscope. After five to seven minutes, worm fragments should have a visibly reduced cuticle and a slurry of cells will be readily visible. Halt the reaction by adding 900 microliters of commercially-available Leibovitz's L-15 medium, supplemented with 10%FBS and penicillin-streptomycin.Centrifuge at 10, 000 times g and at four degrees Celsius for five minutes to pellet isolated fragments and cells. Wash the pelleted cells twice more with cold L-15 supplemented media using one milliliter of media per wash. Resuspend the pelleted cells in one milliliter of L-15 supplemented media, and leave on ice for 30 minutes.Then take the top layer, which is approximately 700 to 800 microliters, and transfer it to a microcentrifuge tube. Use an automated cell counter or hemacytometer to measure the cell density of 10 to 25 microliters of isolated cells. Centrifuge cells from the isolated cell suspension at 10, 000 times g and at four degrees Celsius for five minutes.Discard the supernatant and resuspend the pelleted cells in L-15 supplemented medium to a cell density of no more than six million cells per milliliter to avoid overloading the flow cytometer. Then, sort cells by GFP-positive expression using a flow cytometer capable of sorting samples. GFP-negative cells can be used as a control for non-cell-type-specific analysis.After this, plate the isolated cells into the wells of a sterile six-well plate, place the plate into a plastic container, add a damp wipe or soft tissue paper to add moisture to the cells, and incubate at 20 degrees Celsius. Centrifuge cells from the isolated cell suspension in a 1.5-milliliter RNase-free microcentrifuge tube at 10, 000 times g and at four degrees Celsius for five minutes. Using a micropipette, remove the supernatant and add 100 microliters of M9 medium to wash away the L-15 supplemented medium.Next, centrifuge the cells at 10, 000 times g and at four degrees Celsius for five minutes and remove the supernatant. Repeat this wash and centrifugation process three times total to ensure that all the media is removed, making sure to carefully remove the supernatant each time. Then, add one milliliter of phenol and guanidine isothiocyanate solution to the cells and pipette the suspension up and down carefully.Incubate the mixture at room temperature for five minutes. After this, add 250 microliters of chloroform and gently invert the tube 15 to 20 times. Incubate at room temperature for five minutes.Centrifuge at 10, 000 times g and at 25 degrees Celsius for five minutes. Use a micropipette to carefully remove as much of the top aqueous layer as possible, making sure to not disturb the bottom organic layers. Transfer the bottom layer to a new 1.5 milliliter RNase-free microcentrifuge tube.To the new tube, add 500 microliters of isopropanol and mix by inverting the tube. Incubate this solution at room temperature for five minutes. Then centrifuge the tube at 14, 000 times g and at 25 degrees Celsius for 20 minutes.Place the samples on ice and use a micropipette to remove as much isopropanol as possible. Gently add one milliliter of 70%ethanol in RNase-free double-distilled water to the tube by applying it to the side of the tube. Centrifuge at 10, 000 times g and at 25 degrees Celsius for five minutes.Remove most of the ethanol and let the tube air-dry on ice for three to five minutes. After this, add 15 to 20 microliters of RNase-free double-distilled water. Use a spectrophotometer at 260 nanometers to measure the RNA concentration and purity.In this study, unc-17:GFP-positive cholinergic neurons are extracted and isolated from the roundworm C.elegans for subsequent ex vivo studies. The unc-17 gene is expressed throughout the C.elegans body, and codes for a vesicular acetylcholine transmembrane transporter. The expression of this gene can be observed in head, midbody, and tail neurons, and neuron projections.Representative fluorescence micrographs of unc-17:GFP-positive neurons after isolation, as well as a respective negative control from unmodified N2 wild type animals, are shown here. Isolated cells can stay alive up to three days when supplemented with Leibovitz's L-15 medium and 10%FBS. Representative data of qPCR from GFP-expressing cholinergic neurons, as well as GFP-expressing muscle cells, is shown here.After successfully sorting cells, RNA is isolated via a phenol and guanidine isothiocyanate method, after which cDNA was produced using a synthesis kit. Expression of cholinergic neuron-specific genes, tph-1, a tryptophan hydroxylase, and snb-1, a synaptic vesicle transporter, is elevated in unc-17:GFP isolated cells. However, this expression is not present in myo-3:GFP isolated cells.The inverse is true with the expression of the muscle-specific myosin-heavy chain transcript of myo-2, as the expression of this gene is limited to isolated muscle cells, but not the isolated cholinergic cells. It is important to incubate the worms fully. However, extended incubation of the worm cuticle can result in worm death or additional stress to the animal.Following the isolation, the cells can either be cultured for a period of time, or used for RNA isolation and qRT-PCR analysis. Cultured neurons can be further analyzed by patch clamp electrophoresis measurements. This technique might facilitate ex vivo analysis of specific groups of cells, thereby helping to improve our understanding of cell-specific responses in the context of multicellular organisms at various stages of its lifecycle.As chemicals such as SDS and DTT are used during this experiment, general lab safety procedures should be followed while making and using buffers.

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Developmental Biology

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