The overall goal of this video is to detail the development and implementation of a molecular readout of long-term olfactory adaptation in sea elgan that describes the adaptation state of an animal. This is achieved by tagging a protein kinase G called egg L four with a green fluorescent protein molecule and expressing this protein fusion in the amphi wing cells type C or A WC neurons as a second step animals expressing the GFP tagged form of egg L four in the A WC neurons are cultivated on nematode growth media plates containing the bacterial food source OP 50. Next, after four days, a population of animals are washed off the cultivation plates and exposed to an A WC sensed odor for 80 minutes in order to promote long-term olfactory adaptation.
The results show a nuclear localization of GFP tagged egg L four in the A WC of adapted animals versus a cytoplasmic location of GFP tagged ALE four of unadapted control animals By creating a functional copy of ALE four fields, the two green fluorescent protein. We can track the subcellular localization of ACO four in A WC and describe the animal's behavioral state based upon GFP localization. This molecular tool allows us to rapidly investigate the role of various mutants or treatments in shaping cellular output after sustained neuronal stimulation.
This Protocol begins with the construction of GFP tagged egg L four expressing animals as described in the written procedure accompanying this video. The final integrated line is referred to as I as 500 and the GFP tagged egg L four molecule as GFP egg L four cultivate the IS 500 animals at 25 degrees Celsius on standard 10 centimeter NGM plates. Seated with OP 50 E coli bacteria on day one, pick four to five is 500 animals at larval stage four from plates cultivated at 25 degrees Celsius onto 10 centimeter op 50 e coli seeded plates by using a small platinum wire referred to as a pick to scoop up and to transfer the animals.
Repeat this for four additional plates. Incubate the plates at 25 degrees Celsius on day four. Wash adult populations of IS 500 animals off the large NGM plates by adding as basal buffer to the plate and swirling around gently.
This dislodges the animals from the plate surface and into the buffer solution. Using disposable glass pipettes, aspirate the animals off the plate and transfer them to a 1.5 milliliter einor tube. Begin the translocation assays by washing the is 500 animals in s basil to remove bacteria do not centrifuge.
Animals between washes. Rather allow the animals to settle by gravity in a stationary 1.5 milliliter micro centrifuge tube. It is critical to ensure all bacteria is removed and that NGM plates are free from contamination as residual bacteria will negatively affect the outcome of the translocation assay.
Repeat wash with as basil twice. Make up the adaptation solution by adding 100 milliliters of S basil buffer to a 100 milliliter graduated cylinder. If performing aldehyde adaptation, add 7.5 microliters of aldehyde to 100 milliliters of ESP basil seal the cylinder using a strip of param.
Gently invert the graduated cylinder containing as basil and odor 30 times. To create a uniform emulsion, add animals to each tube trying 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 palate by eye after the animals have settled.
Following S basil wash, remove all liquid from the epi orph tube label one new tube positive and the other new tube as negative. Then add one milliliter of the adaptation mix to the positive adaptation micro centrifuge tube and add one milliliter of S basil to the negative unadapted control micro centrifuge tube. Place a 1.5 milliliter micro centrifuge tube cap protector to each tube and place the tubes on a rotator for 80 minutes.
After 80 minutes, wash the animals three times in S basil, allowing animals to settle by gravity between each wash. Make 2%aros pads containing five millimolar sodium azide by dissolving gel electrophoresis aros. In distilled water, place a single drop of mol and aros containing sodium azide on a glass microscope slide immediately place another glass microscope slide on the first slide to flatten the drop of molten agros.
Leave for 30 seconds and then gently tease apart the microscope slides, leaving one microscope slide with a flat aros pad of approximately one millimeter. At this point, it is imperative that each experimenter devise a key to track which slides are the positive experiment and which slides are the negative control. Then after the worms have settled from the final wash in as basil remove all liquid, then add the animals dropwise onto the aros 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 aros pad by using a small kim wipe.
Place a 0.5 millimeter glass cover slip onto the pad and let stand for two minutes to allow for the sodium azide to immobilize the animals. Next, pass the slides to another person that will blindly score the GFP egg L four localization pattern. This will control for any experiment or bias score between 50 and 100 animals per slide using an upright fluorescent microscope with 10 x 40 x and 63 x magnification objectives and G-F-P-R-F-P filters.
Firstly, locate animals under 10 x magnification using brightfield illumination. Secondly, locate the A WC neuron by turning off the brightfield illumination and using the RFP filter. The promoter odor.
One DS red drives expression in a WB and A WC neurons. The A WC neurons have distinctive oval-shaped cell bodies as compared with a WB cell bodies, which are smaller and circular in shape. Once the A WC cell body has been located, switch to the GFP filter and record the localization of GFP egg L four as nuclear or cytoplasmic.
Using a counter allows the experimenter to keep looking into the eye piece while scoring the slide. This process is repeated at least three times on separate days to produce statistically significant data. An example of the localization pattern of GFP egg L four in the A WC before and after prolonged odor exposure is shown prior to prolonged odor exposure.
GFP egg L four is localized to the cytosol of the A WC after 80 minutes of odor exposure. GFP egg four is localized to the nucleus of the A WC at the behavioral level. Animals with cytosolic GFP egg four in a WC are attracted to a point source of odor.
This graph was generated from representative results from chemotaxis assays of unadapted animals. Note the CHEMOTAXIS index is close to one. Animals exhibiting nuclear G-F-P-E-L four in the A WC are not attracted to a point source of the adapting odor.
This graph was generated from representative results from CHEMOTAXIS assays of adopted animals. Note the CHEMOTAXIS index is close to zero. Once egg L four enters the nucleus of the A WC neurons, it causes stable and long lasting changes in the A WC physiology that persist even when egg L four is no longer in the nucleus.
After 80 minutes of odor exposure, animals will ignore a point source of the adapting odor, and this adaptation will persist even after 120 minutes of recovery. After 80 minutes of odor exposure. GFP Egle four is observed in the nucleus of the A WC.This NU nuclear entry is both necessary and sufficient to induce long-term adaptation in the A WC.After 120 minutes of recovery, these animals no longer exhibit nuclear GFP Eagle four, yet will still ignore a point source of the adapting odor aldehyde at the behavioral level, Not including cultivation times.
Once mastered, this technique can be done in a few hours if it's performed properly.
