STAMs-- weeks 2&3 at the Murphy lab

Richard here, studying learning and memory in C. elegans in Dr. Murphy's lab at Princeton.

For the past two weeks, the primary focus of my research has been to perform short term associative memory training (STAMs) on wild type (N2) and egl-4(ky95) worms.  I may have given a brief description of STAMs in an earlier post, but I'll go into more detail this time:

The experiment starts off with several plates of a strain of worms, which have been bleached so that the worms being tested are all about the same age.  Using M9 buffer, I wash the worms off one of the plates to serve as my naive testing group-- I examine the worms' responsiveness to the chemical butanone without any prior conditioning which will enhance their response.  Then, I wash the rest of the worms off the plates and into a 15 mL tube, where they starve for about an hour.  When this hour has passed, I transfer the worms onto several conditioning plates, with food, and spot a small amount of butanone on each plate so that after an hour of conditioning, the worms will have a developed a strong association between food and butanone.    To test this association, I prepare chemotaxis plates, which look like this:

Chemotaxis assay of wild type right after conditioning-- all the worms are attracted to butanone (left)

The dots on the right and left are both spotted with sodium azide, which stops the worms from moving, and the right and left dots are spotted with ethanol and butanone, respectively.  I put about 100-300 worms on the dot at the bottom, called the origin, and after an hour, I take a picture of the plate and use a digital sorting program to count the worms.  This entire process is called a chemotaxis assay, and I need to perform one at various time points: right after conditioning (0 hr), 30 minutes after conditioning, 1 hr, 2 hr, 4 hr, and 6 hr, using three chemotaxis plates per time point per strain in order to ensure accuracy. The worms being tested at later time points are placed onto hold plates which contain food but no butanone. Factoring in the two hours it takes to starve and then train the worms, the entire experiment lasts 8 hours.  However, there are large chunks of time in between, which gives me time to perform other preparations needed for future STAMs, and time to just relax.

My second week, I performed three STAMs with just the wild type strain, to get acquainted.  My third week, I performed three STAMs with both the wild type strain and the egl-4 strain.  It is known that the egl-4 strain retains its association between food and butanone for several hours, unlike wild type, which loses this association almost completely 2 hrs after conditioning, but during all three of the experiments I performed last week, the egl-4 strain actually displayed even worse chemotaxis towards butanone than did wild type, which is the exact opposite of what was expected.  Two possible explanations immediately come to mind: 1) I am a genius who has just proven the scientific world wrong, or 2) I'm an idiot who, despite having performed the same exact experiment three times, still managed to completely screw up.  Option number 1 sounds  flattering, but is highly unlikely.  Option number 2 seems more realistic, but while there are many things at which I am inept, I am certainly no idiot, never have been, and I am 150% sure that I performed these experiments with as impeccable timing as I could achieve.  With this being said, at this point, I'm not sure what exactly went wrong, but now that my grad student, Geneva, along with the rest of the lab, is back from the International worm meeting at UCLA, hopefully we can figure something out.

In the meantime, I was able to cross my egl-4 mutants with my crh-1 mutants to produce heterozygotes, and then allow those to self fertilize to produce possible double mutants.  In order to verify if any of the offspring chosen are indeed double mutants, I performed PCR and then ran a gel.  However, if there are serious problems with my egl-4 strain, as evidenced by my extremely odd results from my egl-4 STAMs, then this pursuit may be in jeopardy.  All I can do is hope for the best.