Biological Sciences 300/301, Smith College | Neurophysiology
Labs 9-12: Projects on the Crayfish Swimmeret System
http://www.science.smith.edu/departments/NeuroSci/courses/bio330/labs/L9projects.html
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Revised May 7, 2008
View the video: Central Pattern Generator for Crayfish Swimmerets.
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View and listen to recordings of the swimmeret pattern for different concentrations of carbachol. |
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For the remainder of the semester, the laboratory will be devoted to a single experimental project. You will have time to plan, refine, and repeat an experiment. All projects will investigate some aspect of the crayfish swimmeret central pattern generator. We explored potential experiments and some background for the projects in our reading and discussion last week.
Two assignments are associated with the lab project: a one-page abstract and a poster presentation.
Schedule and assignments.
Week 1: Establish a basic preparation to record the swimmeret pattern.
Record from the first roots of the abdominal nerve cord, using either a semi-intact or an isolated nervous system, as described below under Experimental Methods. Try to elicit the swimmeret pattern using an appropriate dose of carbachol or proctolin. If possible, gather your first data.
Weeks 2 -3: Refine and repeat your experiment, modifying your plan if necessary.
Record and analyze data, taking care to show that the results are reproducible. If you are testing the effects of a drug, demonstrate that the effects you see are truly the result of the treatment, and not just a spontaneous response or a sign of deterioration of the preparation. (Usually, being able to wash out the drug and restore the preparation's previous state is a suitable control.) If you are investigating timing or coordination between ganglia, measure and plot your data for each experiment to see if different experiments are consistent.Feel free to check out what other groups are doing, and to offer and receive advice. Working on the same basic preparation allows us to share information and learn from each other.
Week 4: Summarize your experimental work in an abstract and a poster presentation.
ABSTRACT. You and your partner(s) should write an abstract together, with a title and your names at the top. The abstract tells us succinctly what you did and what you found. It is limited to ONE page, single-spaced. You may include diagrams or sample records within that page. Write informatively, for the students who will read your abstract in future years. Please email me a copy of your abstract with embedded figures by 6:00 pm on Monday of the last week of classes (rolivo@email.smith.edu). Abstracts will be printed and distributed to our class and also placed online for future classes.POSTER. Create a poster about your project jointly with your lab partner(s). On your poster, tell us the project's purpose, possibly a little about what is already known, the important aspects of your methods (including pitfalls to watch out for), and what you found out. If you have useful data, we'd like to see selected records or graphs that summarize your results. Posters can be constructed using informal means, such as brown wrapping paper and marking pens (put your effort into the content, not the lettering). You can also use AppleWorks on our lab computers to create panels for your poster. Posters should be ready for viewing one-half hour after the start of our usual lab time. Your group will be asked to give a five-minute talk about your poster to the class.
Posters and abstracts from previous classes are available online*: 2009, 2008, 2007, 2006, 2005. (*Smith campus only)
Some comments on grading. Your experiments, your project abstract, and your poster will all be components in the grade for the laboratory course, along with your portfolio of data from the first part of the semester. Lab projects will be graded on the basis of the sensibility and skill with which the experiments were designed and carried out. It is understood that in some cases, any useful results may represent a triumph. A good project will have been thought through clearly, with data analyzed to demonstrate that responses are repeatable, related to the magnitude of the treatment, and (if possible) reversible. The presentation of the project should be clear, organized, and informative.
Experimental methods.
Video: Central Pattern Generator for Crayfish Swimmerets.
The basic preparation of the crayfish's abdominal nerve cord is the same one we used in Lab 7. Motor neurons send their axons to the swimmeret muscles in root 1 of the second through the fifth abdominal ganglia. Recordings can be made from a first root of a semi-intact preparation using suction electrodes, but you will get better control over drug concentrations if you record from an isolated nerve cord using the pin and vaseline electrodes, described below.
To prepare an isolated nerve cord, follow the procedures outlined in Lab 7 to expose the abdominal nerve cord, and then continue with the dissection shown in the video for this lab. In cutting all the roots, be especially careful to cut the first roots (N1) as far from each ganglion as possible, while you cut the other roots very short. This will avoid confusion about which roots are N1s after the cord is isolated.
On a small dry dissecting dish, make a ring of vaseline that is tall enough to hold back the saline that you will later place in the dish. Move the dissected nerve cord into the dry dish, and drape the branch or branches of a first root through the vaseline so the cut end of the root lies inside the circle. Add a drop or two of saline inside the ring, and create a pool of saline around the main part of the nerve cord. You may also wish to pin out each end of the nerve cord using fine minutenadeln ("tiny needles").
Place pin electrodes in the main pool of saline and in the center of the vaseline ring. The pin and vaseline-ring combination functions like a suction electrode. The vaseline is like the tight-fitting tip of a suction electrode, separating the saline inside the ring from the saline outside. As local circuit currents from action potentials in the axons move through the vaseline region, the wires inside and outside the ring detect a difference in potential. To get good recordings of spikes, it is important that there not be a leak through the vaseline.
To apply drugs, gently remove the main pool of saline around the nerve cord using a transfer pipette. Add some of the new drug solution as a wash, remove it, and add the new drug solution again. The drug will diffuse into each ganglion and reach the synaptic regions in the neuropil. Note that drugs are applied to the main pool surrounding the nerve cord, so that they can diffuse into the ganglia where the synapses are located. Drugs are not placed in the recording wells, where they would reach only the cut ends of axons.
Monitor neural activity on your oscilloscope screen, your audio monitor, and directly on your chart recorder. Rhythmic CPG bursts will be easy to hear, and you will see the bursting pattern easily on the chart recorder or on the oscilloscope at slow sweep speeds. When you have interesting data, you will also want to digitize it on your computer using Chart software. This will later let you zoom in on details of the activity and create records for your poster.
Some helpful hints:
- The pin electrodes have very delicate wires,
so treat them gently. If a wire or pin breaks off, let an
instructor know. They are easy to fix.
- We have fine dissecting scissors that also
need to be treated very gently. No stabbing them into
your dissecting dish in a moment of frustration! Be sure
to wash and try them carefully at the end of each
afternoon.
- Drug solutions are always made up and diluted
in crayfish saline, not distilled water. Again,
note that drugs are applied to the main pool
surrounding the ganglia, not in the recording
wells.
- If you plan to combine two drugs (for example,
proctolin and nicotine), make up your stock solutions of
each individual drug at twice the highest
concentration you plan to use. To test one drug at its
highest concentration (eg, proctolin), dilute your stock
solution 1:1 with saline. To combine the drugs, mix the
two stock solutions in suitable proportions. For example,
mixing the two stock solutions 1:1 will create a new
solution in which each drug's stock concentration has
been diluted in half.
- Work with small volumes. You only need a few
ml to create a pool around the nerve cord, and proctolin
is expensive. The only reason to make more than 5 or 10
ml of a solution is if you need to make a big dilution
(eg, 1:100) of a primary stock solution. See about
joining forces with other groups that are using the same
drugs that you are.
- Avoid test-tube build-up. Resist the
temptation to make a large number of dilutions in advance
-- you may end up wasting many of them. Instead, make
dilutions when you need them (it's a very quick process).
Usually, you can make serial dilutions, which also helps
avoid large volumes. We have calibrated centrifuge tubes
(15 ml and 50 ml) and calibrated transfer pipettes (1 ml)
for making dilutions.
- Label your solutions AND keep them together in a beaker labeled with your group's names and lab day. Label tape and markers are on the front bench.
Links
Supplement: Anatomy of the Crayfish Nervous System.
Lab 8: Discussion of the crayfish swimmeret system.
Appendix: Capturing Oscilloscope Screenshots
Appendix: Using EasyGraf Chart Recorders.
Appendix: Capturing Data with PowerLab and Chart.
Appendix: Screen Shots and AppleWorks Posters.
© 2003 - 2006 by Richard F. Olivo. Permission is granted to non-profit educational institutions to reproduce or adapt this Web page for internal use provided that the original source and copyright are acknowledged.