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.
<ol>
	<li>Prepare and synchronize worms via the bleaching method as described by T. Stiernagle<sup cla...

<ol>
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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 ...

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