Measuring Associative Learning in Chemotaxis of the Nematode Caenorhabditis elegans

One of the most fascinating questions in neuroscience is how learning memory emerge from the plasticity of neurons. Both plasticity and learning decline with age. But it is largely unclear why we investigate the mechanisms responsible for this decline.

Using the nematode model organisms in neuritis elegans. It has become clear that nearly every behavior of C.elegans depends on context and experience, and that they share mechanisms of associative and non associative learning with other organisms. Remarkably key determinants of C.elegans learning such as neural activity and insulin signaling are also implicated in regulating the itching processes of the brain and even the entire organism.

The basic technology is used in studying chemotaxis divided Petri dishes as through microscopes. We also use cameras with tracking software and custom built behavioral chambers for trajectory tracking and calcium imaging with microfluidics to visualize the physiological mechanisms behind the behaviors we observe on the Petri dish. Currently C.elegans sort chemotaxis Assays have been performed on either gradient plates or quadrant plates.

Gradient plates can be demanding to perform because the gradient changes over time and quadrant plates, they have been used to compare two different concentrations of salt only. That's why we introduced the use of white plates for this Assay. We want to understand why the ability to learn, which depends on neural plasticity declines with age.

We aim to use the chemotaxis assay to test whether specific signaling pathways are responsible for this decline. Based on this, we aim to develop treatments targeting this age-related cognitive decline. To begin obtain nematode growth, medium auger plates fed with live escherichia coli OP 50.

Add Caenorhabditis elegans N two on the plate and maintain overnight at 20 degrees Celsius upside down to minimize moisture loss. The next day, transfer 30 young adult Caenorhabditis elegans onto the nematode growth plate. Allow animals to lay eggs for approximately eight hours at 20 degrees Celsius.

After the timed egg laying, remove all adult worms from the plate. Culture the plate upside down at 20 degrees Celsius for three days. For the assay, seed half of the batch of the desired groups of conditioning plates with 200 microliters of OP 50 per plate.

Allow the seeded plates to dry overnight at 20 degrees Celsius in an incubator alongside the unseeded plates. Prepare the desired concentration of one liter sodium chloride solution in a glass bottle. Autoclave the bottles for 15 minutes at 121 degrees Celsius.

Then let the bottles cool for 30 minutes at 55 degrees Celsius in a water bath. Meanwhile, label the bottom of the YP tree dish section with the corresponding sodium chloride concentration, and draw a five millimeter radius circle around the plate center where the walls meet. Place each bottle one at a time on a heated magnetic stirer.

Add 25 milliliters of one molar potassium phosphate solution at pH six to each bottle. Mix thoroughly for two minutes. Then sequentially add calcium chloride cholesterol, and magnesium sulfate while stirring.

Next, aliquot the zero millimolar medium into one compartment of a Y Petri dish until it reaches the top of the internal wall. And let the medium solidify for approximately five minutes. Aliquot the 100 millimolar medium into the second compartment of the Y Petri dish until it touches the solidified zero millimolar section adjacent to it.

Then use a sterile 10 microliter pipette tip to break the surface tension along the y wall and connect it to the zero millimolar sodium chloride section. Let the medium solidify for five minutes. Aliquot the 50 millimolar medium into the remaining compartment of the Y Petri dish until it touches the solidified zero millimolar and 100 millimolar sections.

Then use a sterile 10 microliter pipette tip to break the surface tension along the Y wall and connect the 50 millimolar sodium chloride section to the other two sections. After letting the medium solidify for five minutes, store the plates overnight at 20 degrees Celsius. To begin pipette one milliliter of sterile M nine buffer onto a growth plate containing Caenorhabditis elegans.

Swirl the plate gently to dislodge the worms from the OP 50 bacterial lawn and the auger forming a suspension. Tilt the plate to collect the worm suspension in M nine buffer using a one milliliter plastic pipette tip with the top part cutoff. Transfer the suspension to a 1.5 milliliter micro centrifuge tube.

Allow the worms to settle at the bottom of the micro centrifuge tubes for approximately 60 seconds forming a pellet. Then remove the supernatant using a one milliliter pipette without disturbing the pellet. Wash the pellet with one milliliter of sterile M nine buffer twice.

To remove residual OP 50. Reese suspend the pellet in 300 microliters of M nine buffer. After the final wash.

Swirl the suspension for even distribution of worms and slowly transfer 50 microliters of worm suspension containing approximately 200 to 300 worms to each conditioning plate. For plates seeded with op 50, deposit the worm droplet away from the bacterial food lawn and let it get absorbed into the auger. Keep the plates upside down in an incubator at 20 degrees Celsius for four hours.

After conditioning, pipette one milliliter of sterile M nine buffer onto a conditioning plate and swirl it gently to dislodge the worms forming a suspension. Then tilt the plate to collect the worm suspension using a one milliliter plastic pipette tip and transfer it to a 1.5 milliliter micro centrifuge tube. In 60 seconds, the worms settle down and form a pellet.

After removing the supernatant, wash the worms two times with one milliliter of sterile M nine buffer. Then fill the micro centrifuge tube with M nine buffer up to the one milliliter mark. Once the worm pellet forms remove 960 microliters of the supernatant without disturbing the pellet.

Now swirl or shake the tube gently to resuspend the worms. Transfer 20 microliters of this suspension containing approximately 100 to 150 worms to the center of an assay plate where the y wall segments meet. Let the worms roam for 30 minutes at 20 degrees Celsius.

Then count the number of worms in each section of the assay plate and count them again after 30 minutes. Calculate the Chemotaxis index per plate per time point using the specified formula. Wild type Caenorhabditis elegans behavior was significantly affected by four hour conditioning on plates with different sodium chloride levels with or without food.

After 30 minutes on the assay plate, worms conditioned with high sodium chloride concentrations and food, as well as those conditioned with no sodium chloride and no food showed a positive chemotaxis index indicating a preference for higher sodium chloride concentrations. Conversely, worms conditioned with high sodium chloride concentrations and no food, medium sodium chloride concentrations, and no food and no sodium chloride with food showed a negative chemotaxis index indicating a preference for lower sodium chloride concentrations. The Sodium chloride Food Association persisted after 60 minutes with the Chemotaxis index, often changing in worms conditioned with food indicating dynamic associative learning as animals update this association while roaming and searching for food.