We would like to thank Scott Hamilton, and members of the O'Halloran lab for careful reading of this manuscript. We also thank our anonymous reviewer for excellent suggestions and insightful comments.

1. Construction of GFP Tagged EGL-4 Expressing Animals
<ol>
	<li>Clone the translational fusion GFP::EGL-4 under the promoter for the odr-3 gene (use 2678 bp directly upstream of the start codon): (p)odr-3::GFP::EGL-4. The odr-3 promoter drives expression in the amphid neuron pairs: AWA; AWB; AWC; and weakly in ASH.</li>
	<li>Inject the plasmid (p)odr-3::GFP::EGL-4 into wildtype (N2) animals at 50 ng/&mu;l with the co-injection markers using standard germ-line transformation techniques8: (p)ofm-1::GFP9 (25 ng/&mu;l), and (p)odr-1::dsRed10 (25 ng/&mu;l). The odr-1 promoter drives expression in the AWC and AWB amphid neuron pairs, and the ofm-1 promoter drives expression in the coelomocytes.</li>
	<li>Subject the transgenic animals expressing the extrachromosomal arrays detailed in Step 1.2 to the Ultra Violet (UV)/trimethylpsoralen (TMP) integration protocol11.</li>
	<li>Bleach synchronize the animals12 and cultivate them on Nematode Growth Media (NGM) plates containing OP50 E. coli to the L4 stage. Wash L4 animals off NGM plates with M9 buffer to remove OP50 E. coli bacteria.</li>
	<li>Remove M9 buffer and add 50-100 &mu;l of 30 &mu;g/ml TMP working solution onto the worm pellet in the 1.5 ml microcentrifuge tube. Wrap the 1.5 ml microcentrifuge tube in foil to block ambient light, and agitate gently on a rotator for 15 min in the foil-wrapped 1.5 ml microcentrifuge tube. Transfer the worms in the TMP/worm solution to a large unseeded NGM plate.</li>
	<li>Expose the worms on the NGM plate to UV 350 &mu;J(X100) using a Stratalinker 2400. Then add worms to an OP50 containing NGM plate and incubate in the dark for 5 hr.</li>
	<li>Pick 50 transgenic worms to approximately 10 seeded plates (2-5 worms each plate). Transfer P0's to new plates every 24 hr for 3 days.</li>
	<li>Pick 250 clonal F1 animals (1 animal per plate). Clone out 2-4 F2 animals from each F1 plate. Check F3 animals to look for a 100% transmission line by looking at (p)ofm-1::GFP expression pattern under a dissecting fluorescent microscope.</li>
	<li>Outcross the final integrated line(s) with wildtype N2 animals 5 times. We will refer to the final integrated line as pyIs500, and the GFP-tagged EGL-4 molecule as GFP::EGL-4.</li>
</ol>
2. Cultivation and Maintenance of Animals for Nuclear Translocation Assays
<ol>
	<li>Cultivate the pyIs500 animals at 25 &deg;C on standard 10 cm NGM plates (see below for recipe) seeded with OP50 E. coli bacteria13 (Figure 1).</li>
	<li>On Day 1, pick four to five L4 pyIs500 animals from plates cultivated at 25 &deg;C onto five separate 10 cm OP50 E. coli seeded plates and incubate at 25 &deg;C (that is, 4-5 L4 animals per plate).</li>
	<li>On Day 4, wash adult populations of pyIs500 animals off the large NGM plates using S-Basal buffer (see below for recipe). Use disposable glass pipettes to transfer animals, as they will stick to the side of plastic pipette tips.</li>
</ol>
3. Long-term Odor Induced Nuclear Translocation Assays
<ol>
	<li>Wash the pyIs500 animals 3 times in S-Basal to remove bacteria. Do not centrifuge animals between washes; rather allow the animals to settle by gravity in a stationary 1.5 ml microcentrifuge tube. It is critical to ensure all bacteria is removed, and that NGM pates are free from contamination, as residual bacteria will negatively affect the outcome of the translocation assay. Also, we have found that autoclaving the 1.5 ml microcentrifuge tubes produces a "sticky" property on the interior walls of the tube that causes animals to stick to the sides, and so use non-autoclaved 1.5 ml microcentrifuge tubes.</li>
	<li>While animals are settling between washes, make up the adaptation solution. Add 100 ml of S-Basal buffer to a 100 ml graduating cylinder. Add adapting odor to the S-Basal and seal the cylinder using a strip of Parafilm. If performing benzaldehyde adaptation add 7.5 &mu;l of benzaldehyde to 100 ml of S-Basal, for butanone adaptation add 11 &mu;l of 2-butaone to 100 ml of S-Basal. Gently invert (do not shake) the graduating cylinder containing S-Basal and odor 30 times to create a uniform emulsion.</li>
	<li>After the final wash in S-Basal, allow the animals to settle and remove all liquid. Label one tube "+ve" or "adapt" and the other tube "-ve" or "unadapt". Then add 1 ml of the adaptation mix to the adaptation microcentrifuge tube (+ve), and add 1 ml of S-Basal to the unadapted control microcentrifuge tube (-ve). Try to keep numbers of animals between 100 and 200 in each tube. With some practice, it is possible to estimate the approximate number of animals in a pellet by eye.</li>
	<li>Place a 1.5 ml microcentrifuge tube cap protector to each tube (prevents lids from popping open) and place the tubes on a rotator for 80 min. We have found that odor exposure between 60 min and 80 min yields similar results (both result in long-term adaptation responses). However, 80 min exposure provides more consistent results in our hands. Thus all our long-term adaptation assays are standardized by using an 80 min odor exposure (Figure 2).</li>
	<li>After 80 min, wash animals 3 times in S-Basal. Allow animals to settle by gravity between each wash.</li>
</ol>
4. Short-term Odor-induced Nuclear Translocation Assays
<ol>
	<li>In the case of short-term odor adaptation, use the same protocol as per long-term odor-induced translocation assays (Steps 3.1-3.4), however instead of incubating the pyIs500 animals in adapting odor for 80 min, use a 30 min exposure. The quantification of odor-induced nuclear translocation assays outlined below (Step 5) is the same for long-term and short-term exposures.</li>
</ol>
5. Scoring the Odor-induced Nuclear Translocation Event
<ol>
	<li>Make 2% agarose pads containing 5 mM sodium azide (NaN3) by dissolving gel electrophoresis agarose in dH20. Place a single drop of molten agarose containing NaN3 on a glass microscope slide. Immediately place another glass microscope slide on the first slide to flatten the drop of molten agarose. Leave for 30 sec, and then gently tease apart the microscope slides leaving one microscope slide with a flat agarose pad of ~1 mm.</li>
	<li>After the final wash in S-Basal, allow the worms to settle in the 1.5 ml microcentrifuge tube and remove all liquid. Then add the animals drop-wise onto the agarose pads of several microscope slides. It's important to keep the numbers of animals per slide to between 50 and 100. Too many animals per slide will complicate scoring and often create a scattering glow after blue-light excitation. Excess liquid may be wicked from the agarose pad by using a small Kim-wipe. Place a 0.5 mm glass cover-slip onto the pad and let stand for 2 min to allow for the NaN3 to immobilize the animals. Note, animals can be recovered from the slides after scoring. The animals will recover from the NaN3 anesthetic after ~1 hr if transferred to a seeded NGM plate into a drop of M9 Buffer.</li>
	<li>At this point it is imperative that each experimenter devise a key to track which slide is the experimental (+ve) slide and which slide is the control (-ve) slide, and pass the slides to another person that will blindly score the GFP:EGL-4 localization pattern. This will control for any experimenter bias.</li>
	<li>Score between 50 and 100 animals per slide using an upright fluorescent microscope with 10X, 40X and 63X magnification objectives and GFP/RFP filters. Firstly, locate animals under 10X magnification using bright-field illumination. Secondly, locate the AWC neuron by turning off the bright-field illumination and using the RFP filter. The (p)odr-1::dsRed drives expression in AWB and AWC neurons. The AWC neurons have distinctive oval shaped cell bodies as compared with AWB cell bodies which are smaller and circular in shape (Figure 2).</li>
	<li>Once the AWC cell body has been located, switch to the GFP filter and record the localization of GFP:EGL-4 as nuclear or cytoplasmic. Using a counter allows the experimenter to keep looking into the eyepiece while scoring the slide. Note: Steps 5.1-5.5 are repeated at least 3 times on separate days to produce statistically significant data (Figures 1-2).</li>
</ol>
6. Monitoring GFP-tagged EGL-4 during the Recovery from Long-term Odor Adaptation
<ol>
	<li>After Step 3.5, divide the populations of pyIs500 animals into multiple aliquots and score for nuclear versus cytoplasmic GFP::EGL-4 in the AWC at time point zero (that is, after 80 min exposure) and multiple subsequent time points (Figure 3).</li>
	<li>Transfer both the control (-ve) and experimental (+ve) animals using disposable glass pipettes onto NGM plates.</li>
	<li>Allow animals to recover for various lengths of time and re-examine the localization of GFP::EGL-4in AWC at each time point as in Steps 5.1 to 5.5.</li>
</ol>
7. Materials
NGM plates: For 1 L add to 1 L ddH20: 21 g Bacto-Agar; 3 g Sodium Chloride (NaCl); 2.5 g Bacto-Peptone. Autoclave Media. After autoclaving, cool the agar until the temperature reads 54 &deg;C. Add the following solutions: 25 ml 1M Potassium Phosphate Buffer; 1 ml 1 M Magnesium Sulfate (MgSO4); 1 ml 1 M Calcium Chloride (CaCl2); 1 ml Cholesterol in Ethanol (5 mg/ml stock).
1 M Dibasic K2HPO4 174.2 g/mol: For 500 ml: In 400 ml ddH2O, dissolve 87.1 g Dibasic K2HPO4. Adjust volume to 500 L with ddH2O. Filter sterilize using a 500 ml Sterile Filter Unit.
1 M Monobasic KH2PO4 136.1 g/mol: For 1 L: In 800 ml ddH2O, dissolve 136.1 g Monobasic KH2PO4. Adjust volume to 1 L with ddH2O. Filter sterilize using a 500 ml Sterile Filter Unit.
1 M Potassium Phosphate (K3PO4) Buffer pH 6.0: For 1 L: In a 1 L Sterile Filter Unit, add 868 ml of 1 M Monobasic KH2PO4. Allow unit to filter through, then add 132 ml of 1 M Dibasic K2HPO4. Allow unit to filter all the liquid. Remove filter unit and adjust pH to 6.0. Store at room temperature.
S-Basal: For 1 L: add 50 ml 1M K3PO4 Buffer, pH ~6.0; and 20 ml 5M NaClaq. Fill to 1 L with ddH2O. Autoclave Solution.
M9: For 1 L: 3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O. Fill to 1 L with ddH20. Sterilize by autoclaving.

<ol>
	<li>Colbert, H. A., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-specific+adaptation+pathways+generate+olfactory+plasticity+in+C.+elegans.">Odorant-specific adaptation pathways generate olfactory plasticity in C. elegans.</a> Neuron. 14 (4), 803 (1995).</li><li>Colbert, H. A., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+signals+modulate+olfactory+acuity,+discrimination,+and+memory+in+Caenorhabditis+elegans.">Environmental signals modulate olfactory acuity, discrimination, and memory in Caenorhabditis elegans.</a> Learning and Memory. 4 (2), 179 (1997).</li><li>Bargmann, C. I., 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=Odorant-selective+genes+and+neurons+mediate+olfaction+in+C.+elegans.">Odorant-selective genes and neurons mediate olfaction in C. elegans.</a> Neuron. 74 (3), 515 (1993).</li><li>L'Etoile, N. 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=The+cyclic+GMP-dependent+protein+kinase+EGL-4+regulates+olfactory+adaptation+in+C.+elegans.">The cyclic GMP-dependent protein kinase EGL-4 regulates olfactory adaptation in C. elegans.</a> Neuron. 36, 1079-10 (2002).</li><li>Lee, J. I., 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=Nuclear+entry+of+a+cGMP-dependent+kinase+converts+transient+into+long-lasting+olfactory+adaptation.">Nuclear entry of a cGMP-dependent kinase converts transient into long-lasting olfactory adaptation.</a> PNAS. 107 (13), 6016 (2010).</li><li>O'Halloran, D. M., 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=Regulators+of+AWC-mediated+olfactory+plasticity+in+Caenorhabditis+elegans.">Regulators of AWC-mediated olfactory plasticity in Caenorhabditis elegans.</a> PLoS Genetics. 5 (12), e1000761 (2009).</li><li>O'Halloran, D. M., 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=Changes+in+cGMP+levels+affect+the+localization+of+EGL-4+in+AWC+in+Caenorhabditis+elegans.">Changes in cGMP levels affect the localization of EGL-4 in AWC in Caenorhabditis elegans.</a> PLoS ONE. 7 (2), e31614 (2012).</li><li>Mello, C. C., 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=Efficient+gene+transfer+in+C.elegans:+extrachromosomal+maintenance+and+integration+of+transforming+sequences.">Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences.</a> EMBO Journal. 10, 3959-39 (1991).</li><li>Miyabayashi, 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=Expression+and+function+of+members+of+a+divergent+nuclear+receptor+family+in+Caenorhabditis+elegans.">Expression and function of members of a divergent nuclear receptor family in Caenorhabditis elegans.</a> Dev. Biol. 215, 314 (1999).</li><li>L'Etoile, N. D., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfaction+and+odor+discrimination+are+mediated+by+the+C.+elegans+guanylyl+cyclase+ODR-1.">Olfaction and odor discrimination are mediated by the C. elegans guanylyl cyclase ODR-1.</a> Neuron. 25 (3), 575 (2000).</li><li>Mello, C., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+transformation.">DNA transformation.</a> Methods Cell Biol. 48, 451 (1995).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> Wormbook. , (2006).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77 (1), 71 (1974).</li><li>Kauffman, A., Parsons, L., Stein, G., Wills, A., Kaletsky, R., Murphy, C. <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+Positive+Butanone+Learning,+Short-term,+and+Long-term+Associative+Memory+Assays.">C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays.</a> J. Vis. Exp. (49), e2490 (2011).</li><li>Berkowitz, L. A., Hamamichi, S., Knight, A. L., Harrington, A. J., Caldwell, G. A., Caldwell, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+C.+elegans+Dopamine+Neuron+Degeneration+Assay+for+the+Validation+of+Potential+Parkinson's+Disease+Genes.">Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes.</a> J. Vis. Exp. (17), e835 (2008).</li><li>Pittenger, C., Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+search+of+general+mechanisms+for+long-lasting+plasticity:+Aplysia+and+the+hippocampus.">In search of general mechanisms for long-lasting plasticity: Aplysia and the hippocampus.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 358 (1432), 757 (2003).</li></ol>

No conflicts of interest declared.

The odor-induced nuclear entry of a GFP-tagged EGL-4 molecule described here provides a robust molecular readout of olfactory adaptation in C. elegans. The odor-induced nuclear translocation assays are straightforward and require only a few days of preparation time. The pyIs500 animal that we have constructed for these assays, expresses a marker that illuminates the AWC neuron as well as expressing the GFP-tagged EGL-4 protein. Thus, we feel that an experimenter with little to no experience working with...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments </td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>Difco</td>
			<td>DF0140-07-4</td>
			<td>NGM plates</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Chemical</td>
			<td>S671-10</td>
			<td>NGM plates</td>
		</tr>
		<tr>
			<td>Bacto Peptone</td>
			<td>Difco</td>
			<td>DF0118-07-2</td>
			<td>NGM plates</td>
		</tr>
		<tr>
			<td>Potassium Phosphate Dibasic</td>
			<td>Fisher Chemical</td>
			<td>S375-500</td>
			<td>S-Basal buffer and NGM plates</td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic</td>
			<td>Fisher Chemical</td>
			<td>P285-500</td>
			<td>S-Basal buffer and NGM plates</td>
		</tr>
		<tr>
			<td>Kimwipes - Small</td>
			<td>Kimberly-Clark</td>
			<td>LS2770</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethanol 100%</td>
			<td>Gold Shield Chemical Co.</td>
			<td>43196-115</td>
			<td>diluting odors for chemotaxis assays</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C8106-500G</td>
			<td>NGM plates</td>
		</tr>
		<tr>
			<td>Magnesium Sulphate</td>
			<td>MP Biomedicals</td>
			<td>150136-500G</td>
			<td>NGM plates</td>
		</tr>
		<tr>
			<td>Sodium Azide 99%</td>
			<td>Fisher Scientific</td>
			<td>ICN10289180</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>Agarose - UltraPure</td>
			<td>Invitrogen</td>
			<td>16500-500</td>
			<td>Agarose pads</td>
		</tr>
		<tr>
			<td>Benzaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>B1334-100G</td>
			<td>AWC odor</td>
		</tr>
		<tr>
			<td>Butanone, ACS Grade</td>
			<td>Sigma-Aldrich</td>
			<td>360473-500ML</td>
			<td>AWC odor</td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes - 1.5 ml Colored</td>
			<td>Denville</td>
			<td>LS8147</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pasteur Pipet Disposable Glass 5-3/4"</td>
			<td>Fisher Scientific</td>
			<td>13-678-20B</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stratalinker</td>
			<td>Stratagene</td>
			<td>Stratalinker 2400</td>
			<td>UV integration</td>
		</tr>
		<tr>
			<td>Filter Vacuum Bottle - 500 ml</td>
			<td>Nalgene</td>
			<td>09-740-25B</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a molecular readout of long-term olfactory adaptation in c. elegans

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

An example of the localization pattern of GFP::EGL-4in the AWC before and after prolonged odor exposure is shown in Figure 2. Prior to prolonged odor exposure, GFP::EGL-4 is localized to the cytosol of the AWC (Figure 2B), and after 80 min odor exposure GFP::EGL-4 is localized to the nucleus of the AWC (Figure 2D). At the behavioral level, animals with cytosolic GFP::EGL-4 in AWC are attracted to a point source of odor (Figure 2C graphs representative re...

Watch this Scientific Journal Video about A Molecular Readout of Long-term Olfactory Adaptation in C. elegans at JoVE.com

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

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

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

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

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10.7K Views.  George Washington University. Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

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

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

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

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

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

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

In vivo Laser Axotomy in C. elegans

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

In vivo Laser Axotomy in C. elegans

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

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

Automated Quantification of Synaptic Fluorescence in C. elegans

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

Automated Quantification of Synaptic Fluorescence in C. elegans

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

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

In vivo Neuronal Calcium Imaging in C. elegans

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

In vivo Neuronal Calcium Imaging in C. elegans

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

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

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals&#39; access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal&#39;s body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal&#39;s body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.

We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal&#39;s electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

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 ...

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has proven to be an invaluable model organism in helping to tackle this challenge, from inspiring technological approaches,&#160;building the human brain connectome, to actually shedding light on the specific molecular drivers of basic functional patterns. The bulk of the behavioral studies in C. elegans have been performed on solid substrates. In liquid, animals exhibit behavioral patterns that include movement at a range of speeds in 3D, as well as partial body movements, such as a posterior curl without anterior shape change, which introduce new challenges for quantitation. The steps of a simple procedure, and use of a software that enables high-resolution analysis of C. elegans swim behavior, are presented here. The software, named CeleST, uses a specialized computer program that tracks multiple animals simultaneously and provides novel measures of C. elegans locomotion in liquid (swimming). The measures are mostly grounded in animal posture and based on mathematics used in computer vision and pattern recognition, without computational requirements for threshold cut-offs. The software tool can be used to both assess overall swimming prowess in hundreds of animals from combined small batch trials and to reveal novel phenotypes even in well-characterized genetic mutants. The preparation of specimens for analysis with CeleST is simple and low-tech, enabling wide adaptation by the scientific community. Use of the computational approach described here should therefore contribute to the greater understanding of behavior and behavioral circuits in the C. elegans model.

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

An efficient and simple methodology for computer-based analysis of nematode swimming behavior in liquid is described. The method requires little to no investment for C. elegans laboratories. The hardware used is standard, and the computer software for the behavioral analysis (CeleST) is an open source one.

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its completely mapped nervous system and transparent anatomy, presents an ideal model for understanding real-time neural dynamics using calcium indicators. In combination with microfluidic technologies and experimental designs, calcium-imaging studies using these indicators are performed in both free-moving and trapped animals. However, most previous studies utilizing trapping devices, such as the olfactory chip described in Chronis et al., have devices designed for use in the more common hermaphrodite, as the less common male is both morphologically and structurally dissimilar. An adapted olfactory chip was designed and fabricated for increased efficiency in male neuronal imaging with using young adult animals. A turn was incorporated into the worm loading port to rotate the animals and to allow for the separation of the individual neurons within a bilateral pair in 2D imaging. Worms are exposed to a controlled flow of odorant within the microfluidic device, as described in previous hermaphrodite studies. Calcium transients are then analyzed using the open-source software ImageJ. The procedure described herein should allow for an increased amount of male-based C. elegans calcium imaging studies, deepening our understanding of the mechanisms of sex-specific neuronal signaling.

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of an adapted &#34;olfactory chip&#34; for the efficient calcium imaging of C. elegans males is described here. Studies of male exposure to glycerol and a pheromone are also shown.

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its ...