The overall goal of the following experiment is to test the short and long-term memory of a learned positive association between food and the chemo. Attractant butane, using C elegance as a model. This is achieved by first starving, synchronized, well fed young adult worms for one hour.
Next, the worms are trained in the presence of food and butan known by mast conditioning for short-term associative memory, and by spaced conditioning for long-term associative memory. After conditioning, chemotaxis towards butan known is measured and compared to naive chemotaxis. To study memory retention results are obtained that show the duration of short and long-term associative memory of the learned food Butan known association based on changes in the learning index.
This method can help answer key questions in the cognitive aging and disease fields, such as what genetic pathways are required for neuronal function in normal aging and neurodegenerative disease states, and how learning and memory can be improved. Under conditions of cognitive impairment, Worms are prepared using standard methods on 100 millimeter high growth media plates, seated with one milliliter of OP 50 e coli. Use young adults at a density of around 5, 000 worms or less per plate.
Collect the worms by gently pouring M nine buffer onto the plate and swirling the worms free. Try to minimize agitation, which may dislodge unwanted bacteria. Next, tilt the plate and pull up the buffer worm mixture from the corner.
Using a P 1000 pipe Pepin. Transfer the worms to a 15 milliliter conical tube. Taking care not to pipe pet the worms against the side if worms remain on the plate.
Repeat this Step one to two times. Let the worms settle to the bottom of the tube by gravity. Centrifugation is not recommended for best results.
Once settled, remove the supinate by vacuum and wash with at least three to four milliliters of M nine buffer. Working quickly to prevent starvation. Repeat this procedure two times for a total of three washes.
At this point, a cohort of worms can be set aside to serve as the naive group in the chemotaxis assays that will be demonstrated later to precondition worms for training, add three to four milliliters of M nine buffer to the tube and allow the worms to starve at room temperature for one hour, prepare the conditioning plates by streaking two microliters of 10%butane in 95%Ethanol on the inside of the lids of 60 millimeter NGM plates that have been seeded with 500 microliters of OP 50 e coli. Remove the supinate from the tube of starved worms and use a P 1000 pipe Pepin to transfer 100 to 200 microliters of worms to each conditioning plates. Incubate conditioning plates at room temperature for one hour after conditioning.
Wash the worms from the plates with around one milliliter of M nine buffer and transfer to a 15 milliliter conical tube. Repeat the gentle washing methods demonstrated previously until the buffer appears clear. Once washed, a subset of worms may be removed and tested in the chemotaxis assay to serve as the time zero group for one times mast associative learning to set up plates representing additional time points.
Transfer 100 to 200 microliters of worms to seeded 60 millimeter NGM plates. Incubate these post condition plates for 0.51 or two hours at room temperature. After each time point, gently wash worms from the plates into a 15 milliliter conical tube and one to two times more.
With M nine buffer following the same gentle washing procedures, short-term associative memory is tested using the chemotaxis assay, which will be demonstrated later. Prepare the conditioning plates and worms as demonstrated for short-term training, but allow the worms to incubate at room temperature for only 30 minutes. After incubation, collect the worms as previously demonstrated.
Transfer them to UNSEEDED 60 millimeter NGM plates. Staff the worms on the unseed plates at room temperature for 30 minutes After the worms are starved, wash worms with M nine buffer and place back into the 15 milliliter conical tube. Repeat these steps until a total of seven conditioning blocks and six starve blocks have been completed.
When conditioning is complete, wash the worms from the conditioning plates back into a conical tube. As shown previously, a cohort of worms may be used to test for seven times spaced associative learning immediately after conditioning. Transfer the remaining population of worms to 100 millimeter HGM plates seated with one milliliter of OP 50.
Again, apply 100 to 200 microliters of worms per plate to ensure that there are enough bacteria to support the population. Incubate the post conditioning plates at 20 degrees Celsius for 1624 or 40 hours representing the long-term associative memory time points to test for long-term associative memory of the food butan known association. Gently wash the worms off the plates into a 15 milliliter conical tube, and again, one to two times with M nine buffer after each time point and test the worms in the chemo Texas assay.
To prepare the chemotaxis assay plates, mark the bottom of unseated 100 millimeter NGM plates with spots near the side at three locations as seen here. Spot one microliter of one molar sodium azide at the odorant and control spots. No more than 15 minutes before starting the assay to minimize diffusion of the paralyzing agents.
Next spot, one microliter each of 95%ethanol and 10%butone of the appropriate spots on the marked assay plate. On top of the previously spotted sodium azide. Remove as much of the M nine buffer as possible from the tube of settled worms.
Use a P 20 pipe Pepin with a precut tip to deliver 15 microliters of worms in three five microliter intervals to the origin of the assay plate. Next, twist the corner of a Kim wipe to a small point and use it to blot up the excess M nine buffer. Being careful not to puncture the agar.
Removing excess buffer releases the worms onto the assay plate. If using imaging to count worms, an image of the origin immediately after this release will give the total number of animals for an assay plate. Incubate the chemotaxis assay plate for one hour at room temperature after incubation, count the number of worms at the origin ethanol and butone spots, as well as the total number of worms on the assay plate.
Generally worms paralyzed by sodium azide will be within a one centimeter radius of the spots and will appear stick straight. With many animals stacked against each other, worms can be hand counted or quantified using imaging and analysis software. We prefer the latter for ease and reliability of results.
Once the worms at the origin ethanol and butone spots are quantified, calculate the KeyAx index as seen here. The typical naive chemotaxis index for wild type worms responding to 10%Butone is around 0.2. Mast or spaced training generally increases the CHEMOTAXIS index to 0.7 to 0.8 at times zero to give a learning index of around 0.6.
Naive chemotaxis is highly sensitive to proper worm care. This figure demonstrates the marked difference in the CI between well fed and starved naive worms and highlights the importance of proper technique. Memory retention is reflected in the time it takes for the chemo Texas index to return to naive levels when the learning index equals zero in wild type animals.
Short-term associative memory begins to decline by one hour after mass training lasts about two hours and is independent of the transcription factor kreb by comparison. Long-term associative memory typically does not begin to significantly decline until 16 hours after space training lasts up to 40 hours and is Creb dependent. While attempting this procedure, it is important to remember that chemotaxis assays can be influenced by starvation or excessive odors in the environment, and also that memory and worms can be disrupted by too much physical agitation.
