We wish to acknowledge the cooperative spirit of all Caldwell Lab members. A Basil O&rsquo;Connor Scholar Award from the March of Dimes and a CAREER Award from the National Science Foundation to GAC, as well as an Undergraduate Research Science Program Grant from the Howard Hughes Medical Institute to The University of Alabama, have funded neuronal excitability and epilepsy research in the Caldwell Lab.

Aldicarb Exposure Paradigm
<ol>
<li>On the first day, ensure that at least thirty young adult stage worms of each genotype and of each replicate will be available for aldicarb assays on the second day. It is best if an experimenter selects fifty or more L4 stage worms onto fresh NGM plates (best without nystatin), which contain E. coli (preferably OP50) as a food source, and grow them for 12-24 hours at a consistent and permissive temperature (20&deg;C to 22&deg;C is best, although 25&deg;C is okay). </li>
<li>On the second day, make a 100 mM stock solution of aldicarb with 70% ethanol (EtOH) and 30% ddH2O. Spread the appropriate amount of aldicarb onto NGM minus nystatin plates with defined volumes to achieve the desired aldicarb concentrations. We consistently use 0.5 mM aldicarb by plating 37.5 &mu;L of 100 mM aldicarb onto 7.5 mL NGM plates. Allow the aldicarb plates to dry for roughly 30-60 minutes at room temperature. It is not necessary to crack the lids. Alternatively, aldicarb can be added to NGM and stored at 4&deg;C for one week. &nbsp;</li>
<li>After drying, plate consistent volumes of E. coli (preferably OP50) onto the center of each aldicarb plate and dry for another 30-60 minutes at room temperature. We consistently plate 25 &mu;L of OP50, which creates a sufficiently sized food lawn to keep the worms concentrated in a small spot without overcrowding. &nbsp;</li>
<li>When the food lawn is dry, one may proceed with aldicarb assays. Due to the subjective nature of aldicarb assays, it is highly recommended that experiments be performed "blindly". A colleague of the primary experimenter could re-label the original plates with worms to be assayed. Likewise, the colleague could transfer worms from the original plates to ciphered aldicarb plates immediately before starting a timer. If the experimenter anticipates assaying a particular strain of worms with a characteristic phenotype, such as uncoordination, then there must also be a control with a similar phenotype to reduce bias. Furthermore, it is best if the experimenter assays a wild-type strain, as well as a resistant strain and a hypersensitive strain, in parallel to help standardize experiments. The experimenter should strive to analyze a consistent number of worms for each replicate. We consistently analyze thirty worms of a single genotype for each replicate. We also perform at least three replicates for each experiment. An experienced experimenter should be able to analyze at least six strains at a time. &nbsp;</li>
<li>Count the number of paralyzed worms by prodding in a consistent manner each worm with a platinum wire. We consistently prod our worms twice on the head and twice on the tail every 30 minutes for a total of three hours. Cessation of pharyngeal pumping may also be used to define paralysis, but only if the experimenter employs a consistent definition of paralysis over all assays. Also, it is worth noting that some worms, especially those that are resistant to aldicarb, may attempt to crawl off the plate. In this case, the experimenter may spread a consistent amount of palmitic acid, a physical barrier to worm locomotion, around the aldicarb plates. We spread 25 &mu;L of 10 mg palmitic acid/mL EtOH.</li>
</ol>
PTZ Exposure Paradigm
<ol>
<li>On the first day, ensure that at least thirty young adult stage worms of each genotype and of each replicate will be available for PTZ assays on the second day. It is best if an experimenter selects fifty or more L4 stage worms onto fresh NGM plates (best without nystatin), which contain E. coli (preferably OP50) as a food source, and grow them for 12-24 hours at a consistent and permissive temperature (20&deg;C to 22&deg;C is best, although 25&deg;C is okay). </li>
<li>On the second day, make a 0.5 g PTZ/mL ddH2O stock solution of PTZ. Spread the appropriate amount of PTZ onto NGM minus nystatin plates with defined volumes to achieve the desired PTZ concentrations. Because we prefer dose-response assays for PTZ, we plate varying amounts of PTZ onto 7.5 mL NGM plates. For example, one may plate 37.5 &mu;L of the PTZ stock solution onto a 7.5 mL NGM plate to make a 5 mg PTZ/mL solution. Allow the PTZ plates to dry for roughly 60-120 minutes at room temperature. It is not necessary to crack the lids. Alternatively, PTZ can be added directly to NGM, but it should be immediately used for assays after solidifying. The stability of PTZ is considerably less than that of aldicarb. Therefore, assays with older PTZ plates are less reliable. PTZ should be stored at -20&deg;C in a desiccator with a drying agent before use. We prefer to discard PTZ approximately two months after opening to ensure stability. </li>
<li>After drying, plate consistent volumes of E. coli (preferably OP50) onto the center of each PTZ plate and dry for another 30-60 minutes at room temperature. We consistently plate 25 &mu;L of OP50, which creates a sufficiently sized food lawn to keep the worms concentrated in a small spot without overcrowding. &nbsp;</li>
<li>When the food lawn is dry, one may proceed with PTZ assays. Although PTZ assays are less subjective than aldicarb assays, it is still highly recommended that experiments be performed "blindly". A colleague of the primary experimenter could re-label the original plates with worms to be assayed. Likewise, the colleague could transfer worms from the original plates to ciphered PTZ plates immediately before starting a timer. If the experimenter anticipates assaying a particular strain of worms with a characteristic phenotype, such as uncoordination, then there must also be a control with a similar phenotype to reduce bias. Furthermore, it is best if the experimenter assays a wild-type strain, as well as strains with anterior or full-body convulsions, in parallel to help standardize experiments. The experimenter should strive to analyze a consistent number of worms for each replicate. We consistently analyze thirty worms of a single genotype for each replicate. We also perform at least three replicates for each experiment. An experienced experimenter should be able to analyze at least three strains at a time. Yet, the frequency and intensity of PTZ-induced convulsions usually peak between 15-30 minutes of exposure, and then attenuate as worms become paralyzed or, in some cases, acclimate to the drug. </li>
<li>Count the number of "epileptic-like" convulsing worms every 30 minutes for a total of one hour. It is best to score the number of worms with anterior convulsions, which we call "head-bobs", separately from the number of worms with full-body convulsions, full-body paralysis, which we call "tonic", or a combination of anterior convulsions with BWM paralysis, which we call "tonic-clonic". Most worms of a single genotype exhibit only one kind of convulsion. However, tonic-clonic worms often become completely tonic, so one should continue the assay past 30 minutes, even if all worms have already exhibited convulsions. Also, it is worth noting that some worms may attempt to crawl off the plate. In this case, the experimenter may spread a consistent amount of palmitic acid around the PTZ plates. We spread 25 &mu;L of 10 mg palmitic acid/mL EtOH.</li>
</ol>
&nbsp;
Behavioral Responses of Selected C. elegans Synaptic Transmission Mutants to Aldicarb and PTZ
<table style="height: 157px;" border="1">
<tbody>
<tr>
<td>Mutant Name</td><td>Synaptic Role</td><td>Behavior without Drug</td><td>Behavioral Response to Aldicarb (compared to wild-type N2)</td><td>Behavioral Response to PTZ</td>
</tr>
<tr>
<td>tom-1(ok188)</td>
<td>inhibits synaptic transmission</td>
<td>uncoordinated</td>
<td>enhanced rate of paralysis</td>
<td>indistinguishable from wild-type</td>
</tr>
<tr>
<td>unc-43(n498n1186)</td>
<td>complex</td>
<td>uncoordinated</td>
<td>enhanced rate of paralysis</td>
<td>full-body convulsions</td>
</tr>
<tr>
<td>unc-25(e156)</td>
<td>promotes GABA transmission</td>
<td>uncoordinated</td>
<td>enhanced rate of paralysis</td>
<td>anterior convulsions, full-body paralysis</td>
</tr>
<tr>
<td>snb-1(md247)</td>
<td>promotes synaptic transmission</td>
<td>uncoordinated</td>
<td>reduced rate of paralysis</td>
<td>anterior convulsions</td>
</tr>
<tr>
<td>unc-4(e120)</td>
<td>promotes ACh transmission</td>
<td>uncoordinated</td>
<td>reduced rate of paralysis</td>
<td>indistinguishable from wild-type</td>
</tr>
</tbody>
</table>
Two neural stimulants, an acetylcholinesterase inhibitor, called aldicarb, and a GABA receptor antagonist, called pentylenetetrazole (PTZ), can be used in a complementary manner to characterize C. elegans synaptic transmission mutants. Excess excitatory acetylcholine (ACh) accumulates at worm body wall neuromuscular junctions (NMJs) from deleterious mutations in negative regulators of ACh transmission (e.g. tom-1 and unc-43) or positive regulators of inhibitory GABA transmission (e.g., unc-25). Conversely, excitatory ACh levels at worm NMJs are diminished by deleterious mutations in positive regulators of general synaptic transmission (e.g., snb-1) or ACh-specific transmission genes (e.g. unc-4). When compared to wild-type N2 worms, mutant worms with elevated excitatory ACh transmission at NMJs exhibit enhanced rates of aldicarb-induced paralysis, whereas mutant worms with lowered excitatory ACh transmission demonstrate reduced rates of aldicarb-induced paralysis. Although PTZ disrupts neuronal synchrony at C. elegans body wall muscles, not unlike aldicarb, PTZ also antagonizes inhibitory GABA at C. elegans head muscles. As a result, aldicarb sensitivity cannot accurately predict PTZ sensitivity. Mutant worms with specific defects in negative or positive regulation of ACh transmission are indistinguishable from wild-type N2 worms in the presence of PTZ, whereas mutant worms with defects in positive regulation of general synaptic transmission or specific defects in inhibitory GABA transmission exhibit robust PTZ-induced anterior convulsions. Moreover, unc-43 loss-of-function mutants display full-body convulsions in the presence of PTZ and likely have additional synaptic transmission abnormalities, which contribute to their unique drug responses.

<ol>
	<li>Mahoney, T. R., Luo, S., Nonet, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+synaptic+transmission+in+Caenorhabditis+elegans+using+an+aldicarb-sensitivity+assay.">Analysis of synaptic transmission in Caenorhabditis elegans using an aldicarb-sensitivity assay.</a> Nat. Protoc. 1, 1772-1777 (2006).</li><li>Williams, S. N., Locke, C. J., Braden, A. L., Caldwell, K. A., Caldwell, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epileptic-like+convulsions+associated+with+LIS-1+in+the+cytoskeletal+control+of+neurotransmitter+signaling+in+Caenorhabditis+elegans.">Epileptic-like convulsions associated with LIS-1 in the cytoskeletal control of neurotransmitter signaling in Caenorhabditis elegans.</a> Hum. Mol. Genet.. 13, 2043-2059 (2004).</li></ol>

Current protocols for aldicarb exposure with C. elegans do not allow experimenters to distinguish between mutants with specific deficits in ACh transmission and mutants with generalized deficits in synaptic transmission, as both classes of mutants exhibit resistance to aldicarb. Likewise, aldicarb cannot be used to determine if mutants have specific deficits in GABA transmission or generalized failures to negatively regulate ACh transmission, as both classes of mutants exhibit hypersensitivity to aldicarb. Resul...

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		<th>Material Name</th>
		<th>Type</th>
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<td>Aldicarb</td>
<td>Reagent</td>
<td>Supelco, Inc.</td>
<td>PS734</td>
<td>Purchasable from Sigma</td>
</tr>
<tr>
<td>Pentylenetetrazole</td>
<td>Reagent</td>
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paradigms for pharmacological characterization of c. elegans synaptic transmission mutants

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the anatomy and connectivity of its nervous system has been determined from electron micrographs and refined by pharmacological assays. In this video, we describe how two complementary neural stimulants, an acetylcholinesterase inhibitor, called aldicarb, and a gamma-aminobutyric acid (GABA) receptor antagonist, called pentylenetetrazole (PTZ), may be employed to specifically characterize signaling at C. elegans neuromuscular junctions (NMJs) and facilitate our understanding of antagonistic neural circuits.
Of 302 C. elegans neurons, nineteen GABAergic D-type motor neurons innervate body wall muscles (BWMs), while four GABAergic neurons, called RMEs, innervate head muscles. Conversely, thirty-nine motor neurons express the excitatory neurotransmitter, acetylcholine (ACh), and antagonize GABA transmission at BWMs to coordinate locomotion. The antagonistic nature of GABAergic and cholinergic motor neurons at body wall NMJs was initially determined by laser ablation and later buttressed by aldicarb exposure. Acute aldicarb exposure results in a time-course or dose-responsive paralysis in wild-type worms. Yet, loss of excitatory ACh transmission confers resistance to aldicarb, as less ACh accumulates at worm NMJs, leading to less stimulation of BWMs. Resistance to aldicarb may be observed with ACh-specific or general synaptic function mutants. Consistent with antagonistic GABA and ACh transmission, loss of GABA transmission, or a failure to negatively regulate ACh release, confers hypersensitivity to aldicarb. Although aldicarb exposure has led to the isolation of numerous worm homologs of neurotransmission genes, aldicarb exposure alone cannot efficiently determine prevailing roles for genes and pathways in specific C. elegans motor neurons. For this purpose, we have introduced a complementary experimental approach, which uses PTZ.
Neurotransmission mutants display clear phenotypes, distinct from aldicarb-induced paralysis, in response to PTZ. Wild-type worms, as well as mutants with specific inabilities to release or receive ACh, do not show apparent sensitivity to PTZ. However, GABA mutants, as well as general synaptic function mutants, display anterior convulsions in a time-course or dose-responsive manner. Mutants that cannot negatively regulate general neurotransmitter release and, thus, secrete excessive amounts of ACh onto BWMs, become paralyzed on PTZ. The PTZ-induced phenotypes of discrete mutant classes indicate that a complementary approach with aldicarb and PTZ exposure paradigms in C. elegans may accelerate our understanding of neurotransmission. Moreover, videos demonstrating how we perform pharmacological assays should establish consistent methods for C. elegans research.

Watch this Scientific Journal Video about Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants at JoVE.com

Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants

This video demonstrates how to employ two neural stimulants, aldicarb and pentylenetetrazole (PTZ), in complementary ways to study synaptic function in the nematode, C. elegans. This complementary approach may also be used to shed light on evolutionarily conserved mechanisms for modulating neuronal synchrony and has implications for epilepsy and seizures.

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the ...

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14.1K Aufrufe.  University of Alabama. Dieses Video zeigt, wie zwei neuronalen Stimulanzien, Aldicarb und Pentylentetrazol (PTZ), in komplementärer Weise zu synaptische Funktion in dem Fadenwurm C. elegans Studie beschäftigen. Mit diesem ergänzenden Ansatz kann auch verwendet werden, um Licht auf evolutionär konservierte Mechanismen zur Modulation neuronaler Synchronität vergossen werden und hat Auswirkungen auf Epilepsie und Krampfanfällen.

Serie: Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that exhibit partial and variable transmission rates of extrachromosomal arrays to the next generation. In addition, transgenic animals are mosaic and express the transgene in a variable number of cells. Extrachromosomal arrays can be integrated into the C. elegans genome using UV irradiation to establish nonmosaic transgenic strains with 100% transmission rate of the transgene. To that extent, F1 progenies of UV irradiated transgenic animals are screened for animals carrying a heterozygous integration of the transgene, which leads to a 75% Mendelian transmission rate to the F2 progeny. One of the challenges of this method is to distinguish between the percentage of transgene transmission in a population before (X% transgenic animals) and after integration (&#8805;75% transgenic F2 animals). Thus, this method requires choosing a nonintegrated transgenic line with a percentage of transgenic animals that is significantly lower than the Mendelian segregation of 75%. Consequently, nonintegrated transgenic lines with an extrachromosomal array transmission rate to the next generation &#8804;60% are usually preferred for integration, and transgene integration in highly transmitting strains is difficult. Here we show that the efficiency of extrachromosomal arrays integration into the genome is increased when using highly transmitting transgenic lines (&#8805;80%). The described protocol allows for easy selection of several independent lines with homozygous transgene integration into the genome after UV irradiation of transgenic worms exhibiting a high rate of extrachromosomal array transmission. Furthermore, this method is quite fast and low material consuming. The possibility of rapidly generating different lines that express a particular integrated transgene is of great interest for studies focusing on gene expression pattern and regulation, protein localization, and overexpression, as well as for the development of subcellular markers.

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

This protocol describes a rapid and low material consuming procedure for the integration of transgenic extrachromosomal arrays into the Caenorhabditis elegans genome using ultra violet (UV) irradiation. Furthermore, this protocol is particularly well suited for transgenic lines that transmit extrachromosomal arrays at a high rate.

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound responses and repair in this model have illuminated our understanding of the cytoskeletal and genomic responses to tissue damage. The two most commonly used methods to wound the C. elegans adult skin are pricks with microinjection needles, and local laser irradiation. Needle wounding locally disrupts the cuticle, epidermis, and associated extracellular matrix, and may also damage internal tissues. Laser irradiation results in more localized damage. Wounding triggers a succession of readily assayed responses including elevated epidermal Ca2+ (seconds-minutes), formation and closure of an actin-containing ring at the wound site (1-2 hr), elevated transcription of antimicrobial peptide genes (2-24 hr), and scar formation. Essentially all wild type adult animals survive wounding, whereas mutants defective in wound repair or other responses show decreased survival. Detailed protocols for needle and laser wounding, and assays for quantitation and visualization of wound responses and repair processes (Ca dynamics, actin dynamics, antimicrobial peptide induction, and survival) are presented.

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity.

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...