Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise

The overall goal of this report is to be a teaching tool for advanced high school and undergraduate students in physiology courses that participate in physiological experimentation. Students learn to expose and record contractions in the crayfish hind gut. A further step is to excise the hind gut from the animal, which allows direct application of compounds.

Next, the force and rhythm of hindgut smooth muscle contractions are monitored while applying various pharmacological agents to examine the mechanisms that alter the strength of contraction. Results are obtained that show the action of modulators on the neural inners or directly on muscle based on pharmacological assays and receptor subtyping. Hello, I'm Dr.J Mercier of the Department of Biological Sciences at Brock University.

I've been using the crayfish hind gut as a preparation for conducting research on neurotransmitters that control visceral muscles. I've also been using this preparation as a teaching tool and as part of a collaboration with Dr.Robin Cooper's lab at the University of Kentucky, we're perfecting a number of student run experiments for teaching purposes. This method can provide insight into the function of the crayfish hang gut.

It can also be applied in studies of disease such as diseases involving in peristaltic rhythms. It can also be used to describe the mechanisms underlining the modulation of muscle and neural innervation. Students will learn to interpret physiological and pharmacological data through the use of pharmacological agents, neurotransmitters, and neuro hormones, and will acquire a general understanding of receptor function.

With regard to agonists and antagonists, students will construct dose response curves and will present data in graphical form for statistical analysis To anesthetize the animal, place a crayfish measuring six to 10 centimeters in body length on ice for five to 10 minutes. Hold the anesthetized crayfish from behind the claws with one hand. Quickly cut from the eye socket to the middle of the head on both sides, and then behead the crayfish.

The blood from the preparation will be sticky when it tries, so wash the tools when completed. Next, cut off the legs and the claws. Then the left and right tail fins, leaving the middle tail fin cut along the dorsal side of the abdomen until the last segment is reached.

Then cut along the last segment and down the other side. Take off the dorsal shell. Place the dissected crayfish into the sigar lined Petri dish.

Poor crayfish saline. To cover the preparation used dissection pins to secure the preparation to the Petri dish. The hind gut is usually surrounded by a mass of blood vessels and connective tissue.

Remove these with forceps. Taking care not to damage the gut. The hind gut is now visible along the abdomen.

Allow the dissected crayfish preparation to sit in the saline solution for about 10 minutes so it can recover from the shock of dissection and saline exposure. Watch for the slow peristaltic contractions of the hind gut. To begin, the rate of contractions can now be calculated.

Count the number of contractions that occur in 30 seconds and record it in a log. Also record the type of contractions that occur and whether they are peristaltic or spastic. Note the direction of any peristaltic waves.

Count the rate of contractions close to the anus and midway along the abdomen, see if there are rate differences between these two regions. Have a variety of testing solutions ready and at the same temperature as the bathing saline. The first solution tested here will be one micromolar serotonin made in crayfish saline.

Using a pipette, remove the saline from the cavity of the cray fish's exposed abdomen and apply the one micromolar serotonin solution directly onto the hind gut. Immediately after adding the new solution, wait 30 seconds and then count 30 seconds worth of contractions in the region near the anus, and then count the next 30 seconds worth of contractions midway along the abdomen. Record these numbers in a chart and also note the type of contractions that occur.Next.

Pour out the test solution from the recording dish and rinse the hind gut several times with normal crayfish saline. Then rinse the preparation every 30 seconds for five minutes. At this point, you can add the next test solution, which in this demonstration is one Micromolar Topamine.

Record your observations as before and repeat this process for the remainder of the test compounds. When using multiple concentrations of the same substance, be sure to start with the lowest concentration and work to higher concentrations. The setup to measure the force of the GI contractions includes a laptop with lab chart seven software, the bridge pod, the power lab, 26 T, and the force transducer.

Before you can measure the force of the GI muscle contractions, you need to isolate the GI tract. To accomplish this, cut away the muscles in the last abdominal segment, place two or more pins through the soft ventral cuticle on either side of the anus, then cut away the rest of the abdomen, leaving the hind gut and anus attached to a small piece of cuticle. The gut should be cut close to the junction of the thorax and the abdomen.

Now move the GI tract to a shallow dish with a sard coated bottom. There is a blood vessel that runs along the dorsal aspect of the GI tract. Carefully pull this away from the GI tract.

Pour fresh crayfish saline onto the preparation. Move the force transducer over to the preparation. Look closely at the pin that is glued to the bottom of the force transducer.

The end that hooks to the gut has a small bend. Hook the small bend of the pin to the cut end of hind gut. After hooking the hind gut, move the dish slightly until the hind gut is taut.

Be careful with this step as the gut should be taut, but not too tight. Being too tight could damage the muscle. When the dish is in the optimal position, use wax or clay to hold the dish steady.

Next, start the software. To begin recording, open the lab chart software. Close the lab chart, welcome center.

Go to setup at the top left. Click on channel settings at the bottom left. Change the channel settings to have only one channel.

Click okay at the right hand of the screen. Go to channel one open bridge pod. Set the range to the most sensitive setting, 200 microvolts in this case at the top of the chart in the right hand corner.

Set cycles per second to 2K. You are now ready to record. Click start to ensure that the setup is working correctly.

Lightly touch the transducer tip with a pipette. You should see a change in force recorded by the software. Click stop on the software, then click start and let the program run.

For about 20 seconds, click stop and add a comment labeled saline. Only calculate the rate of contractions by measuring the time from the beginning of one deflection to the beginning of the next, or by counting the total deflection over a minute or two. Calculate the rate as contractions per minute or as contractions per second depending on how rapidly they are occurring.

To measure the force of contractions, use a relative measure to compare contractions under different conditions on the saved file. Move the marker M to the baseline, then move the cursor to the peak of the deflection and note the value listed on the top right of the screen. This is a delta value that is the difference from M to the peak.

Note that the cursor needs to be kept stationary at the peak in order to measure the change. Then move the M marker and the cursor to the next wave form of interest and repeat the measure to examine the effect of modulators. On this preparation, slowly switch the saline to a test solution.

In this case, one micromolar serotonin, and continue recording the rate and force of contractions as the last test. For this preparation test whether gap junction inhibitors change the rate or force of contractions, gap junction inhibitors include one hep ethanol or saline that has been bubbled with carbon dioxide. These compounds are used last as they may damage the muscle fibers after prolonged exposure to compare the effect of the various compounds, graph the data on a chart.

Another interesting use of this preparation is to record the force generated by the circular muscles. However, if you applied gap junction inhibitors to your preparation, the inhibitors may have damaged the circular muscles, in which case you should use a fresh hind gut preparation to test the circular muscles. To expose the circular muscles, slit the tube of the gut in a longitudinal plane with fine scissors.

Use fine pins to pin one side securely to the dish. Attach the hooked pin on the transducer to the other side of the strip. Expose the preparation to the same compounds that were tested on the longitudinal muscles, the relationship between the compounds tested and the number and type of contractions are shown here.

You can graph the number of contractions versus time for the saline, serotonin, and glutamate solutions. You can graph the number of contractions versus the force of the contractions in millivolts for the saline serotonin glutamate solutions. These graft can be done for both the longitudinal and the circular muscles to test whether the longitudinal and circular muscles in the different regions of the gut exhibit different responses to the various modulators.

The above experiments can be repeated on different regions from the anus and up along the abdomen. While attempting this procedure, it is important to remember to be careful to not damage the GI tract with the tweezers and to not pull too strongly with the force transducer Following this procedure. Other methods like electrophysiological, recordings of the muscle can be performed to address other issues such as ion channel functions and modulations, and which can serve as a model and other tissues in other animal models.