Learning & Memory

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

Method detailed in:

Kauffman, A., Parsons, L., Stein, G., Wills, A., Kaletsky, R., and Murphy, C.T.C. elegans positive butanone learning, short-term, and long-term associative memory assays. Journal of Visualized Experiments (2011) March 11; (49): e2490.

Graphical Abstract

Training

Training

Chemotaxis Assay

Chemotaxis

Preparation of Animals for Assay

  1. Cultivate worms on 100 mm high growth media (HGM) plates seeded with 1 mL OP50 E. coli using standard methods.
  2. Hypochlorite-treat at least two densely populated HGM plates of gravid adult worms to obtain a large synchronized population of eggs.
    Note: For best yields, bleach the first generation to obtain a highly synchronized population, then bleach the second generation as Day 2 adults to obtain eggs for the subsequent step.
  3. Evenly divide eggs onto 3-4 HGM plates seeded with 1 mL OP50 E. coli for each hypochlorite treated plate.
    Note: It is important that the worms are cultivated with plenty of fresh food as starvation can affect the outcome of olfactory assays.
  4. Incubate worms at 20oC for about 72 hours, the time it takes for animals reach the young adult stage.

Pre-conditioning Starve

  1. Double check your HGM plates with young adults to make sure the worms still have plenty of food, and that there are enough worms for the assay (≥ 200 worms per assay plate).
  2. Wash worms off of HGM plates with M9 buffer into a 15 mL conical tube. For the least agitation during washes, gently apply the M9 buffer by pouring a few milliliters onto the HGM plate, and gently swirl the plate to free worms from the sticky OP50 bacteria. Next, tilt the plate, pull up the M9 buffer/worm mixture from the corner of the plate using a P1000 pipetman, and transfer the worms to the 15 mL conical tube. If worms are still left on the plate, repeat this step 1-2X.
    Note: Rough washing dislodges bacteria off of the plate that can make it into the tube with the worms. This bacteria is impossible to get rid of and can interfere with the assays. DO NOT pipette worms against the side of the tube, as this can damage the worms and interfere with the assays.
  3. Let worms settle by gravity (DO NOT centrifuge). Remove supernatant by vacuum and wash with at least 3-4 mL of M9. Repeat 2X for a total of 3 washes.
    Note: Centrifuging during washes will pull down any remaining bacteria into the worm population, which can interfere with the assays. Instead, gently add M9 buffer for subsequent washes by pouring it directly into the 15 mL conical tube. Worms settled at the bottom of the tube should become mixed in the M9 buffer upon its addition. If worms remained settled, gently invert the tube to mix.
  4. After the third wash, use some of the worm population for naíve chemotaxis assays.
    Note: It is critical to work fairly quickly at all wash steps, as worms will begin to starve if they sit too long in M9 buffer, which can increase the naíve chemotaxis toward butanone.
  5. Add 3-4 mL of M9 buffer to the 15 mL conical tube, let worms starve in M9 buffer at room temperature for 1 hour.

Short-term Associative Memory (massed) Training

  1. At the end of the starve period in M9 buffer, streak 2 μL of 10% butanone (in 95% EtOH) on the inside of the lids of 60 mm nematode growth media (NGM) plates that have been seeded with 500 μL OP50 E. coli.
    Note: A good starting population for short- and long-term memory assays is ≥ 500 μL of worms. Multiple conditioning plates are needed per genotype to support the entire population during training. When working with volatile chemicals, only open lids of plates when necessary, leaving them closed at all other times.
  2. Remove the M9 buffer supernatant from the 15 mL conical tube of starved worms by vacuum and use a P1000 pipetman to apply 100-200 μL of worms to the conditioning plates.
    Note: Try to remove as much M9 liquid as possible, so that worms can quickly get to the OP50 food source when transferred to the conditioning plates. Worms will stick to the inside of the pipette tip and can be conserved by pipetting smaller volumes.
  3. Incubate conditioning plates at room temperature for 1 hour.
  4. Wash worms off of plates with ~1 mL M9 buffer into a 15 mL conical tube. Let worms settle by gravity. Wash again 1-2X until M9 buffer in the tube is clear.
    Note: Use the gentle washing methods described. The same 15 mL conical tube can be used from the starve.
  5. After the final wash, remove the M9 buffer supernatant by vacuum, and use some of the worm population for chemotaxis assays to test for 1x (massed) associative learning of the food-butanone association immediately after conditioning (0 minute time point).
    Learning Index (LI) = CI0 hr - CINaive.
  6. Transfer the remaining population of worms to 60 mm NGM plates seeded with 500 μL of OP50 (hold plates). Again, apply 100-200 μL of worms per plate to ensure there are enough bacteria to support the population.
    Note: The number of hold plates used per genotype is at least the number of short-term associative memory time points to be tested.
  7. Incubate post-conditioning plates for 0.5, 1, or 2 hours (short-term associative memory time points) at room temperature .
    Note: Short-term associative memory of wild-type animals ends by 2 hrs after training. Time points may need to be adjusted for different genotypes or conditions.
  8. To test for short-term associative memory of the food-butanone association, after each time point, gently wash worms off plates into a 15 mL conical tube and again 1-2X with M9 buffer. Use worms for chemotaxis assays. Learning Index (LI) = CITime point - CINaive.
    Note: Use the gentle washing methods. For each time point, discard any extra worms that are not used in chemotaxis assays.

Long-term Associative Memory (Spaced) Training

  1. Follow steps 3.1-3.2 in the Short-term Associative Memory assay.
  2. Incubate conditioning plates at room temperature for 30 minutes.
  3. Follow step 3.4 in the Short-term Associative Memory assay.
    Note: To remain inside a 30-minute timeframe, one can begin all washes during training at ~25 minutes.
  4. After the final wash, remove M9 supernatant by vacuum and transfer worms to unseeded 60 mm NGM plates.
    Note: The use of multiple plates per genotype is not required at this step since the worms are being starved.
  5. Starve on 60 mm NGM plates at room temperature for 30 minutes.
  6. Wash worms with M9 buffer into the 15 mL conical tube. Let worms settle by gravity (DO NOT centrifuge).
    Note: While there should be no bacteria in the buffer at this point, centrifuging can be a potentially damaging treatment of the worms, and may disrupt long-term memory formation.
  7. Repeat steps 4.1-4.6 several times until a total of 7 conditioning blocks and 6 starve blocks have been completed. (See Table 1 for a representative time sheet.)
  8. Gently wash worms off plates into the 15 mL conical tube and again 1-2X with M9 buffer.
  9. After the final wash, use some of the worm population for chemotaxis assays to test for 7x (spaced) associative learning of the food-butanone association immediately after conditioning (0 hour time point). Learning Index (LI) = CI0 hr - CINaive.
  10. Transfer the remaining population of worms to 100 mm HGM plates seeded with 1 mL of OP50 (hold plates). Again, apply 100-200 μL of worms per plate to ensure there are enough bacteria to support the population.
    Note: The number of hold plates used per genotype is at least the number of long-term associative memory time points to be tested. 100 mm NGM plates can also be used as post-conditioning plates, but one must be very careful that there is enough OP50 to support the population of worms for at least 16 hours.
  11. Incubate post-conditioning plates for 16, 24, and 40 hours (long-term associative memory time points) at 20oC.
    Note: Long-term associative memory of wild-type animals ends by 40 hrs after training. Time points may need to be adjusted for different genotypes or conditions.
  12. To test for long-term associative memory of the food-butanone association, gently wash worms off plates into a 15 mL conical tube and again 1-2X with M9 buffer after each time point. Use worms for chemotaxis assays. Learning Index (LI) = CITime point - CINaive.
    Note: Use the gentle washing methods. For each time point, discard any extra worms not used in the chemotaxis assays.

Chemotaxis Assay

  1. Prepare the chemotaxis assay plates. Mark the bottom of unseeded 100 mm NGM plates with spots on the bottom and each side of the plate.
    Note: At least 3 replicates per genotype must be run to obtain statistically significant results.
  2. Spot 1 μL of 1 M NaN3 at the odorant and control spot.
    Note: Do not spot your plates with this paralyzing agent more than 15 minutes before starting the assay, or the NaN3 will diffuse away from the spots, and animals will become paralyzed before reaching the odorant or control spots. Be careful not to puncture the agar when applying NaN3 since worms will burrow in punctured areas.
  3. While the worms are settling in the conical tube after several M9 buffer washes, spot 1 μL each of 95% EtOH and 10% butanone at the appropriate spots on the marked assay plate on top of the previously spotted NaN 3.
    Note: Chemicals must be spotted prior to adding the worms. Make a note of which spot has EtOH or butanone. When working with these volatile chemicals, be careful to only open the lids of plates when necessary, and keep them closed at all other times.
  4. Remove as much of the M9 buffer as possible from the tube of settled worms. Use a P20 pipetman with a pre-cut tip to deliver 3X 5 μL worms (200-400 animals) to the origin of the marked assay plate.
    Note: Keep remaining worms in the 15 mL conical tube for use in Pre-conditioning Starve and memory assays. Worms will stick inside pipette tips, so adding worms to the plate in smaller quantities conserves your worm population and reduces the amount of liquid that gets released with the worms onto the assay plate.
  5. Twist the corner of a KimWipe to a small point and use it to blot up the excess M9 buffer. This will release the worms onto the assay plate.
    Note: Be careful not to puncture the agar with the KimWipe, otherwise worms will burrow at the origin. Some worms may be lost in this step as they are pulled into the KimWipe with the M9 buffer when blotting. If using imaging and analysis to count worms, take plates to imaging station before releasing worms from the origin. An image of the worms at the origin immediately after release will give the total number of animals for an assay plate.
  6. Incubate the chemotaxis assay plate for 1 hour at room temperature.
  7. Count the number of worms at origin, EtOH, and butanone spots, as well as the total number of worms on the assay plate.
    Note: Generally, worms paralyzed by NaN3 will be within a ~1 cm radius of the spots, and will appear stick-straight with many animals stacked against each other. If using imaging and analysis software to count worms, take images of the origin, EtOH, and butanone spots. An image of the total amount of worms should have been taken. Worms can be also be "hand-counted."
  8. Calculate the "Chemotaxis Index" (CI). CI = ([(nButanone)-(nEtOH)]/[(Total-nOrigin)].

Worm Counting by Image Analysis

  1. Take high-contrast black-and-white images of worms on chemotaxis assay plates. To calculate a chemotaxis index, images are needed at the butanone, EtOH, and origin spots of a chemotaxis assay plate at the end of an assay, as well as the total number of worms (picture of worms at origin immediately after they are released from M9 buffer at the beginning of the assay.
    Note: Images of the chemotaxis assay plates covering about a 2 cm radius around each spot generally capture all of the animals for each spot.
  2. Make sure the files for all trials of one time point (example Naive, 0 hr, etc.) are contained in one folder, named appropriately.
  3. Files for each chemotaxis plate must be named as such: but#.png (butanone spot, trial #), ori#.png (origin, trial #), eth#.png (ethanol spot, trial #), tot#.png (total, trial #). (For example, if two trials were run for a time point, in the same folder the following files would be present: but1.png, but2.png, eth1.png, eth2.png, ori1.png, ori2.png, tot1.png, tot2.png)
  4. Open Matlab.
  5. In the "Current Directory" line at the top of the Matlab window, browse for folders, and choose the "count_worms_v0.5.3" folder (M-files available as Supplementary Information).
  6. In the "Command Window", type "count_worms_directory()." Hit Enter.
    Note: The default worm size when for this command is min 10, max 80 pixels. To adjust this based on a specific genotype or developmental stage, type "count_worms_directory ('minsize',10,'maxsize', 80)" and change the pixel numbers accordingly.
  7. When prompted, choose the folder of images to be analyzed.
    Note: Only one folder of images can be analyzed at a time.
  8. Each image in the folder being analyzed will pop up with a threshold already set, and particles selected. Drag the rectangle tool over selected particles that are not worms to delete them. When finished with the image, hit the Esc key.
  9. After all images in the folder are checked, 4 columns will appear in the Command Window: (1) image name (example but1); (2) always "[1];" (3) average pixel size for one worm calculated by the program the particular image; and (4) the number of worms calculated to be in the image.
  10. Once this program has run, it will deposit two .csv files (can be opened as Excel spreadsheets) into the folder of images it has just analyzed. The "worm_counts_stats" file provides the output columns found in the Command Window. The "worm_counts_summary" file provides 5 columns: (1) trial number; number of worms in (2) but, (3) eth, and (4) ori spots; and (5) total number of worms.

Materials Notes

  1. To prepare 100 mm high growth media (HGM) plates: Dissolve 3 g NaCl, 20 g Bactopeptone, and 30 g Bacto-agar in 700 mL distilled water. Bring volume to 1 L with distilled water. After autoclaving, cool agar to 65oC and add 4 mL of 5 mg/mL cholesterol in ethanol, 1 mL each of 1 M CaCl2, 1 M MgSO4, and 25 mL of 1 M KPO4(pH = 6.0).
  2. 95% EtOH is used, as higher percentage EtOH tends to contain impurities from the purification process that can affect results. It is a good idea to make the 10% butanone (in 95% EtOH) stock up fresh every few times the assay is run.
  3. P20 tips must have a few millimeters cut off to create a hole large enough for worms to fit through without being damaged. P1000 tips already have holes large enough for the worms to fit through.
  4. The chemotaxis assay plate imaging station in the Murphy lab contains a Firewire camera with a variable magnification lens attached to a boom stand. A backlight is placed at the bottom of the stand underneath a custom imaging platform with a glass top, which allows chemotaxis assay plates to be illuminated from the bottom. "Measurement and Automation" software (National Instruments) is used to capture images. Materials for this imaging station set up are similar to those previously published, and can be found in the Table of specific reagents and equipment.

Representative Results:

A typical naïve chemotaxis index (CI) for wild-type worms for 10% butanone is around 0.2. Massed (1x) or spaced (7x) training generally increases the chemotaxis index to 0.7-0.8 at time 0 to give a learning index (LI = trained CI - naíve CI) of ~0.6. The learning that occurs with massed or spaced training is very robust. A LI of less than 0.5 typically arises when there are problems with the naíve chemotaxis assay, and the animals have a naíve CI of 0.3 or higher. Usually, this occurs because worms are starving or too crowded on cultivation plates between bleaching and the start of the assay, or worms sit too long in M9 buffer between washes and begin to starve; environmental odors can also increase the naíve CI. Starved worms have a much higher naíve CI for 10% butanone. We find that problems with the naíve chemotaxis are generally resolved with improved worm care and culturing.

The duration of memory in these assays is the time it takes for chemotaxis index immediately after training to return to naíve levels, when the learning index = 0. In wild-type animals, short-term associative memory typically begins to decline by 1 hour after massed training, lasts about 2 hours, and is independent of the transcription factor CREB . Long-term associative memory typically does not begin to significantly decline until 16 hours after spaced training, lasts up to 40 hours, and is CREB-dependent. Long-term associative memory may not reach its full potential in several instances: (1) worms do not have enough OP50 E. coli during 30-minute conditioning periods; (2) bacteria remain in the M9 buffer during starvation periods; or (3) worms are damaged during the assay (e.g. worms are pipetted against the side of the tube).

Results are generally statistically significant when 4 or more chemotaxis assay trials are run per genotype per time point. Running six replicates per genotype typically yields very significant results without becoming too much to handle; however, beginners may want to start with only three replicates. Generally, working with 2-3 genotypes or conditions at a time is comfortable for those who are experienced with these learning and memory assays.

Hand-counting worms on chemotaxis assay plates is very time consuming, can add variability between experiments or lab mates, and may also introduce biases when analyzing and interpreting data. Worm counting by image analysis standardizes data collection and analysis across the lab, and on average, cuts data analysis time for experienced lab members to 1/5 of that needed for hand counting. When comparing chemotaxis indices calculated using manual worm counts to those determined by the Count_worms software, we find an average error of 3.07 (± 1.19)%.

Day 1
Time Step
9:00 1 hr starve in M9 buffer
Start naíve chemotaxis assay
10:00 Condition #1 (60 mm NGM plates with food and butanone), 30 min
End naíve chemotaxis assay
10:30 Starve #1 (60 mm NGM plates), 30 min
11:00 Condition #2, 30 min
11:30 Starve #2, 30 min
12:00 Condition #3, 30 min
12:30 Starve #3, 30 min
1:00 Condition #4, 30 min
1:30 Starve #4, 30 min
2:00 Condition #5, 30 min
2:30 Starve #5, 30 min
3:00 Condition #6, 30 min
3:30 Starve #6, 30 min
4:00 Condition #7, 30 min
4:30 Begin 0-hr chemotaxis assay
Transfer remaining worms to hold plates for 16-40 hrs
5:30 End 0-hr chemotaxis assay
Day 2
Time Step
8:30 Begin 16-hr chemotaxis assay
9:30 End 16-hr chemotaxis assay

Table 1. Representative schedule for long-term associative memory assay. In this example, the assay begins on Day 1 at 9 am with a 1-hr starve. 30-min condition and starve periods are alternated until the worms have been conditioned seven times. Chemotaxis assays are run at the beginning (naíve) and end (0 hr) of training. The total run-time from start to finish is 8.5 hours. Worms on hold plates are tested for chemotaxis to butanone starting on Day 2, 16-40 hours after training.

Detailed STAM Protocol

Preparations of animals for assay:

  1. Cultivate worms on 100 mm high growth media (HGM) plates seeded with 1 mL OP50 E. coli.

    Avoid starvation as this will affect the worm’s behavior

    Do not dry your plates in the hood as this might dry out your plates and affect the worms’ behavior

  2. Hypochlorite-treat two to four densely populated HGM plates containing gravid adults. Divide the obtained eggs onto three to four seeded HGM plates and incubate at 20⁰C for at least 72 hours. 

    Use large orifice tips when handling the worms

    Work quickly

Day prior to the assay

  1. Place the chemotaxis plates at room temperature. You will need twenty five nematode growth medium (NGM) plates per genotype.
  2. Seed five NGM plates (your conditioning (2) and hold plates (3)) with 1 mL OP50 E. coli.

    The seeded bacteria on the plates might need more than one day to dry, in this case, plates should be seeded earlier.

Morning before the assay

  1. Prepare 500 mL of a 10% butanone solution in ethanol and vortex

    Use filter tips

  2. Put 500 ml of NaN3 (1 M) in 1.5 ml vial
  3. Put 500 ml of Ethanol in 1.5 ml vial

    Use filter tips

  4. Label your plates, chemotaxis plates, tubes and vials

    Chemotaxis plates should be labeled as depicted here:

    Chemotaxis Plates

  5. Check your plates and make sure you have a) enough worms (4 well-populated plates per genotype), b) your worms are not starved and c) that the worms are at the young adult stage

    Pre- conditioning starve

  6. Put one of the four plates plate aside (for assaying naïve chemotaxis) and wash worms off of your plates with M9 buffer into a 15 mL conical tube.

    Gently apply the M9 buffer to your plates (by pouring or using a squirt bottle)

    Gently swirl your plate to free the worms from the bacteria

    Tilt your plate and pour the M9 buffer/worm mixture into a 15 mL conical tube

  7. Let the worms settle by gravity, remove the supernatant by vacuum and wash 3 more times with 4 mL M9 buffer.

    Do not centrifuge

    Make sure settled worms become mixed when adding M9 buffer. IF not, gently invert the tube

  8. Add 4 mL M9 buffer to the 15 mL conical tube and let the worms starve in M9 buffer for 1 hour at room temperature.

NOTE: it is recommended to test not more than three genotype at the same time. It is however possible to work in parallel, but six genotypes (A, B & C and D, E & F) should be the limit. In this case, the following scheme can be used:

Assay steps

genotype

pre-conditioning starve

A, B & C

naïve chemotaxis

D, E & F

pre-conditioning starve

A, B & C

naïve chemotaxis

D, E & F

STAM training part 1

A, B & C

STAM training part 1

D, E & F

STAM training part 2

A, B & C

STAM training part 2

D, E & F

STAM training part 3 & 4

A, B, C, D, E & F

Naïve chemotaxis assay

  1. Remove the condensation from the chemotaxis plates and spot 1 μL of 1 M NaN3 at the odorant and control spot.
  2. Wash worms off of the conditioning plates with 1 mL M9 buffer into a 1.5 mL microcentrifuge tube using a P1000 pipetman and let worms settle by gravity.

    Use the same gently washing methods as described earlier

    Use large orifice tips

    Tilt your plate to pull of the M9 buffer/worm mixture

  3. Wash the pellet with M9 buffer and let worms settle by gravity
  4. While the worms are settling, spot 1 μL of 95% ethanol at the control sport.
  5. Wash the pellet with M9 buffer and let worms settle by gravity
  6. While the worms are settling, spot 1 μL of 10% butanone at the odorant sport
  7. Remove the supernatant by vacuum

    Remove as much M9 as possible

  8. Using a P20 pipetman, apply 5 μL of the worm pellet onto the origin spot of the chemotaxis plates.

    Use large orifice tips

    Go to the middle of the pellet when pulling up the M9 buffer/worm mixture

    Make sure you pulled up enough worms; if not, discard these worms and use the remaining worms

  9. Twist a piece of KimWipe to a small point and use it to remove excess fluid on the origin spot. Incubate for 1 hour at room temperature and proceed to part 4.

    Gently tap the drop of worms to remove the excess fluid

    Do not try to remove all the excess fluid as this might affects the worms’ chemotaxis

    Keep the chemotaxis plates in a closed container

    Short-term associative memory training

    Part 1

  1. Remove the condensation on the lids of the conditioning plates (NGM plates seeded with 1 mL E. coli OP50) at the end of the starvation period.
  2. Make three streaks on the inside of the lids of the conditioning plates with 2 mL of 10% butanone.

    Per genotype, at least two conditioning plates are required to support the entire population during the conditioning phase

    Close the lids as quick as possible after applying the butanone

    This should be performed right before applying the worms onto the conditioning plates

    The depicted pattern allows the butanone to be evenly distributed throughout the plate

    Butanone Pattern

  3. Remove as much M9 buffer as possible from the 15 mL conical tube of starved worms by vacuum.  Using a P20 pipetman, apply 100 – 150 mL of the worm pellet onto two (or more) condition plates.

    The total volume of worms should be distributed over at least 2 plates

    Too many worms on one conditioning plate might cause starvation. Therefore, one should make sure to properly distribute the worm population

    Remove as much M9 as possible as this will result in less liquid on the plate and therefore allow the worms to reach the OP50 food source quickly

  4. Incbuate at room temperature for 1 hour

    Parafilm the conditioning plates

    Conditioning plates can be kept in a closed container to avoid varying environmental conditions to affect the worms’ behavior

  5. As an additional control, ‘part 1’ can be performed without the use of butanone. As such, mock behavior can be assessed allowing to test whether the worm population is being stressed during the course of the assay. Next, parts 2, 3 and 4 are performed as described below.

Part 2

  1. The condensation is removed from the lids of the hold plates (T30, T60 and T120) and T0 chemotaxis plates at the end of the conditioning period

    For the chemotaxis assay, at least three replicates per genotype must be run but five replicates are recommended (per timepoint)

    T0, T30, T60 and T120 are indicative for the following timepoints:

    T0: 1x associative (massed) learning

    T30:                30 minutes short-term associative memory timepoint

    T60:                60 minutes short-term associative memory timepoint

    T120:              120 minutes short-term associative memory timepoint

  2. Spot 1 μL of 1 M NaN3 at the odorant and control spot.
  3. Wash worms off of conditioning plates with 3 mL M9 buffer into a 15 mL conical tube and let worms settle by gravity.

    Use the same gently washing methods as described earlier

  4. Wash the pellet with M9 buffer and let worms settle by gravity
  5. While the worms are settling, spot 1 μL of 95% ethanol at the control sport.
  6. Wash the pellet with M9 buffer and let worms settle by gravity
  7. While the worms are settling, spot 1 μL of 10% butanone at the odorant sport
  8. Remove the supernatant by vacuum

    Remove as much M9 as possible

  9. Transfer approximately 50 μL of the worm pellet onto each hold plate (T30, T60 and T120; NGM plates seeded with 1 mL E. coli OP50) and put the plates aside

    Hold plates are kept aside for 30 minutes, 1 hour or 2 hours after which the plates are used in part 3

    The hold plates can be kept in a closed container to avoid varying environmental conditions to affect the worms’s behavior

    Use large orifice tips

    Go to the middle of the pellet when pulling up the M9 buffer/worm mixture

    Divide the 50 μL over the entire plate by placing little drops on the plate

  10. Using a P20 pipetman, apply 5 μL of the worm pellet onto the origin spot of the chemotaxis plates.

    Use large orifice tips

    Go to the middle of the pellet when pulling up the M9 buffer/worm mixture

    Make sure you pulled up enough worms; if not, discard these worms and use the remaining worms

  11. Twist a piece of KimWipe to a small point and use it to remove excess fluid on the origin spot. Incubate for 1 hour at room temperature and proceed to part 4.

    Gently tap the drop of worms to remove the excess fluid

    Do not try to remove all the excess fluid as this might affects the worms’ chemotaxis

    Keep the chemotaxis plates in a closed container

    Part 3

  1. After a short-term associative memory training of 30 minutes, 1 hour or 2 hours; the T30, T60 or T120 hold plates, respectively, are used for assessing chemotaxis after a certain period of time on hold plates.

    For the chemotaxis assay, at least three replicates per genotype must be run but five replicates are recommended (per timepoint)

    T0, T30, T60 and T120 are indicative for the following timepoints:

    T0: 1x associative (massed) learning

    T30:                30 minutes short-term associative memory timepoint

    T60:                60 minutes short-term associative memory timepoint

    T120:              120 minutes short-term associative memory timepoint

  2. Remove the condensation from the chemotaxis plates and spot 1 μL of 1 M NaN3 at the odorant and control spot.
  3. Wash worms off of conditioning plates with 1 mL M9 buffer into a 1.5 mL microcentrifuge tube using a P1000 pipetman and let worms settle by gravity.

    Use the same gently washing methods as described earlier

    Use large orifice tips

    Tilt your plate to pull of the M9 buffer/worm mixture

  4. Wash the pellet with M9 buffer and let worms settle by gravity
  5. While the worms are settling, spot 1 μL of 95% ethanol at the control sport.
  6. Wash the pellet with M9 buffer and let worms settle by gravity
  7. While the worms are settling, spot 1 μL of 10% butanone at the odorant sport
  8. Remove the supernatant by vacuum

    Remove as much M9 as possible

  9. Using a P20 pipetman, apply 5 μL of the worm pellet onto the origin spot of the chemotaxis plates.

    Use large orifice tips

    Go to the middle of the pellet when pulling up the M9 buffer/worm mixture

    Make sure you pulled up enough worms; if not, discard these worms and use the remaining worms

  10. Twist a piece of KimWipe to a small point and use it to remove excess fluid on the origin spot. Incubate for 1 hour at room temperature and proceed to part 4.

    Gently tap the drop of worms to remove the excess fluid

    Do not try to remove all the excess fluid as this might affects the worms’ chemotaxis

    Keep the chemotaxis plates in a closed container

    Part 4

  11. Count the number of worms within 1 cm of the control spot, odorant spot and origin and count the total number of worms on the assay plate.
  12. Calculate the chemotaxis index: CI = ([(nButanone)-(nEtOH)]/[(Total-nOrigin)]

Technical considerations and common pitfalls

  1. It is key to have a large starting population (about 500 ml) as this will allow to work quickly without being concerned about losing worms along the way.
  2. Use large orifice tips when handling worms, using normal tips will cause stress and/or damage them.
  3. The used chemicals are volatile, therefore, make sure to keep the lids closed as much as possible and parafilm your plates during the conditioning period.
  4. Do not pipet up and down before pulling up the M9 buffer/worm mixture. Instead, go to the middle of the pellet and pull up the M9 buffer/worm mixture.
  5. When washing worms off HGM plate, make sure to gently apply the M9 to the plate and gently swirl it in order to avoid dislodging bacteria off the plate.
  6. In between the different steps of the assay, plates (conditioning plates and hold plates) can be kept in a closed container in order to avoid varying environmental conditions to affect the worm’s behavior
  7. When testing the mock and conditioned worms simultaneously, make sure to keep both conditions far apart from one another.
  8. Worm should be let to settle by gravity, do not centrifuge.
  9. Do not pipette worms against the side of the tube, as this can damage the worms and interfere with the assays
  10. Try to remove as much M9 liquid as possible, so that worms can quickly get to the OP50 food source when transferred to the conditioning plates. Worms will stick to the inside of the pipette tip and can be conserved by pipetting smaller volumes.
  11. All washing steps should be performed as quick as possible (but with consideration of potential stressful manipulations) in order to avoid starvation while sitting in the M9 buffer.
  12. Working quickly is also important during the chemotaxis assay. Additionally, it is recommended to test not more than three genotype at the same time. It is however possible to work in parallel, but six genotypes should be the limit.
  13. When studying cognitive decline, washes should be reduced to a minimum. Ideally, late L4’s are washed off HGM plates and distributed over fresh plates while ensuring that worms which will be tested at day 3 of day 5 of adulthood will have enough food for 3 or 5 days, respectively.