INTRODUCTION

Endogenous oscillators are fundamental in an organism's routine functioning. In addition to controlling internal rhythms essential for normal physiological survival, they allow an animal to predict the occurrence of relevant environmental stimuli so that it can orient its behavior effectively. Thus, it is not surprising that timing systems based on internal oscillators have evolved in such a diversity of species.

Caenorhabdititis (C.)elegans, a simple invertebrate with only 302 neurons, maintain several behaviors on precisely timed intervals. Behaviors such as pharyngeal pumping, egg laying, locomotive wave patterns, spontaneous reversals, and defecation all occur in defined cycles.

Defecation is an ideal oscillatory behavior to study as it consists of three very distinct motor steps that are easily identifiable and can be scored with certainty. In normal wild-type worms in food, defecation occurs every 45 seconds. This does not greatly vary across individuals as there is a standard deviation of only 3 seconds (Thomas, 1990). The defecation begins with the contraction of the posterior body muscles (pBoc) which propels the contents of the gut anteriorly. Following relaxation of these muscles the anterior body muscles contract (aBoc) driving the pharynx back pressurizing the gut lumen, In the final step a set of muscles in the anus contract (Exp) to expel the gut contents. There have been many mutants isolated that have a defective cycle periodicity. These mutants may allow researchers to genetically define the basic nature of the biological clock.

It is likely that an endogenous oscillator controls the defecation motor program. One of the most striking pieces of evidence for this is that animals that spontaneously stop feeding, cease defecating and when defecation is resumed, it continues in phase with the interrupted cycle. This indicates that the oscillator continues to run even when the behavior is not being expressed. Thomas (1990) found that touch stimuli reset the defecation cycle to zero with head touches being more reliable than tail touches. This resetting occurred regardless of when in the cycle the touch was administered.

Studies of behavioral plasticity in C.elegans have utilized another mechanosensory stimulus, in which a mechanical stimulus creates a vibration of the agar substrate. This is called a tap stimulus. The tap is a more reliable stimulus than touch as it's intensity is objectively controlled from one tap to the next. Wicks and Rankin (Wicks and Rankin, 1995) have determined the neural circuit underlying the response to tap. In studies of habituation Rankin and Colleagues have noted a high level of variability in responses both within and between individual worms. All of the subjects are genetically and developmental identical. They are raised in analogous conditions and are tested according to precise protocol. Keeping in mind the uniformity of the stimulus, this diversity is difficult to justify. One hypothesis is that the timing of the taps in relation to some endogenous cycle are affecting to the worms' response. It may be that understanding the interaction between the cycle and the response to tap is one of the crucial variables that predict how the worm is to respond in any given situation.

Two primary questions that will be tested in these experiments arise from this hypothesis. The first is whether or not tap resets the defecation cycle as does touch and if it does, does this effect habituate. The second question is how the response magnitude of the worm varies across discrete phases of the cycle.

MATERIALS AND METHODS

Subjects: C.elegans Bristol (N2) 4 day old hermaphrodites will be used. Worms were cultured in E.coli (OP50) on nematode growth medium at 20.

Apparatus: All testing was conducted on petri plates filled with 10 ml NGM agar streaked with E.coli under a stereomicroscope Wild Leitz M3Z. The procedure was videotaped using Panasonic camera D5000, Panasonic 1950 VCR, NEC color monitor. A time-date generator (Panasonic 814) superimposed a stop-watch onto the image for timing cycles and stimulus delivery. For testing the petri dish was mounted on a rod attached to a micromanipulator (MM33) so that the plate could be moved smoothly while keeping the worm within the viewer. A mechanical tapper was also mounted on the holder. This tapper consisted of an electromagnetic relay and wire arm. The relay was connected to a Grass 88 stimulator to regulate stimulus delivery. The stimulator was set to deliver a 25ms pulse to this relay.

Procedure: Subjects will be transferred at the young adult stage from original colony plates to streaked plates approximately 24 hours prior to experimentation. They will be incubated at 20 until 2 hours prior to testing time where they will be removed from the incubator and kept at room temperature. Subjects will then placed directly in the testing apparatus where they will be left for 10 minutes before any observations are made. After the 10 minute delay, defecation intervals will be timed until a cycle period is determined; usually five defecation cycles will be recorded before any taps are given.

Experiment 1: A single tap will be administered at various times during the cycle. The defecation interval will be timed, and the cycle allowed to reestablish itself before another tap is given. This will be repeated four times. There will be four groups of worms.

Based on a defecation cycle of 50 seconds, groups will receive taps at either 10 seconds, 20 seconds, 30 seconds or 40 seconds into the defecation cycle. Following the tap the cycles will be observed and recorded. The post tap cycles will be compared pre and post tap as well as between groups that received the tap at different phases of the cycle to determine whether the tap resets the defecation cycle. In addition the magnitude of responses to tap will be compared across groups to determine whether temporal placement of the tap in relation to the cycle affects response magnitude.

Experiment 2: If tap influences the defecation cycle it will be interesting to see whether this effect habituates with repeated stimulation. If tap does not reset the cycle, we will study the effects of the placement of the tap within the cycle to response magnitude. Protocol will be established based on the results of experiment 1.

REFERENCES

Hall JG (1995) Tripping Along the Trail to the Molecular Mechanisms of Biological Clocks Trends in Neuroscience 18: 230-240

Liu D, Thomas JH, Iwasaki K Genes that Control a Temperature Compensated Ultradian Clock in C.elegans

Liu DWC, Thomas JH (1994) Regulation of a Periodic Motor Program in C.elegans Journal of Neuroscience 14(4): 1953-1962

Thomas JH (1990) Genetic Analysis of Defecation in C.elegans Genetics 124:855-872

Wong A, Boutis P, Hekimi S (1995) Mutation in the clk-1 Gene of Caenorhabdititis Elegans Affect Developmental and Behavioral Timing Genetics 139: 1247-1259