Biological Sciences 300/301, Smith College | Neurophysiology
Lab 8: Discussion of the crayfish swimmeret system.
http://www.science.smith.edu/departments/NeuroSci/courses/bio330/labs/L8discussn.html
REVISED: May 7, 2008
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N1 motor neurons
backfilled with cobalt.
(Mulloney &
Hall,
J Comp Neurol. 2000)
Click to enlarge.
Cross
section of N1 showing bundles of motor and sensory
axons.
View the video: Swimmeret Movements
Listen to a
recording from the first roots (N1)
of the third (A3, top trace) and fourth (A4,
bottom
trace) abdominal ganglia after activation of
the
swimmeret motor pattern by 50 µM
carbachol.
In place of an experiment, this week's laboratory will involve a discussion and a writing assignment based on five articles or excerpts about the crayfish swimmeret pattern generator. The discussion will help us design a class project for the remaining weeks of the semester.
The crayfish swimmeret pattern generator is an example of a network of neurons that creates a behavioral pattern, the rhythmic movement of the swimmerets. Each abdominal ganglion contains clusters of motor neurons on the left side and the right side that innervate the power stroke (PS) and return stroke (RS) muscles of the swimmeret on that side. The cluster of motor neurons is located near the base of the first root, N1, in a region called the lateral neuropil (LN), as shown in the micrograph at the left and the diagram below. The motor neuron axons leave the ganglion in the first root, which splits into an anterior branch that goes to the return-stroke muscles (RS), and a posterior branch to the power-stroke muscles (PS). Recording from the two branches of N1 reveals alternating bursts of spikes in the axons of the return-stroke and power-stroke motor neurons. These bursts are responsible for the alternating forward and backward movements of the swimmerets.
Source: Mulloney B, Skinner FK, Namba H, Hall WM (1998) Intersegmental coordination of swimmeret movements: mathematical models and neural circuits. Ann N Y Acad Sci 860:266-80
The RS and PS motor neurons in each LN cluster are in turn driven by four local non-spiking interneurons that form a pattern-generating module. The modules on each side of the ganglion are linked to each other and to adjacent ganglia to coordinate the swimmerets in different segments. In addition, five command interneurons are known that run the length of the cord and send branches to each LN cluster to activate the swimmeret modules.
A diagram of the proposed pattern generating circuit is shown in the next figure. The RSE and PSE excitor motor neurons are shown at the left. These excitatory motor neurons release glutamate at the muscle cells, causing them to depolarize and contract. There are about 30 excitatory motor neurons in each group (RS and PS). Like other crustacean muscles, the swimmeret muscles also receive inhibitory innervation, and the inhibitors are shown in the diagram (PSI and RSI). Inhibitory motor neurons release GABA at the muscle cells, opposing depolarization and reducing contraction. When the excitors to one class of muscles are active, often the inhibitor to the antagonist muscle is also active.
In the pattern generating circuit, the 1A and 1B local interneurons inhibit the PSE and RSI motor neurons, and inhibit their opponent 2A interneurons. The 2A interneurons inhibit the RSE and PSI motor neurons, and also inhibit the opposing 1A and 1B interneurons. The circuit is an example of reciprocal inhibition. The local interneurons also excite spiking coordinating interneurons that fire simultaneously with the return stroke (DSC, descending to the next posterior ganglion) or the power stroke (ASC, ascending to the next anterior ganglion). (Spiking neurons that coordinate the left and right modules in the same ganglion are not shown.) It is not known what excites the motor neurons, since only inhibitory connections to motor neurons have been discovered from the CPG local interneurons.
At the far right, coordinating and command neurons from other segments are shown. A non-spiking interneuron (IN) receives excitatory synapses from ascending (ASC) and descending (DSC) neurons from adjacent ganglia, and drives the local interneurons. Command neurons extending through the nerve cord activate the swimmeret CPG in each ganglion. Three (or four) of the command neurons contain proctolin (PROCT), and one (or two) do not. At the bottom left, sensory input from non-spiking stretch receptors and other sensory receptors connects (in an undefined way) to the motor neurons and local interneurons. It is likely that some of these sensory neurons release ACh as their transmitter.
Model of the proposed circuit for the swimmeret
central pattern generator.
Adapted from Mulloney, B (2003) During fictive locomotion,
graded synaptic currents drive bursts of impulses in
swimmeret motor neurons. J Neuroscience 23(13):
5953-5962, Figure 10.
In experiments, the motor pattern can be recorded from the first roots of each ganglion using suction or pin electrodes. The system is pharmacologically excitable by adding the acetylcholine mimic carbachol or the peptide proctolin to the bath, as you will see from the readings below. It is not yet certain where those drugs act on the swimmeret system, although (as mentioned) some command neurons contain proctolin, and motor neurons (and probably the local interneurons) respond to acetylcholine.
Readings
Five readings will be distributed in class to serve as the basis for our discussion. Additional anatomical information is included in our Web appendix on Crayfish Neuroanatomy.
1. Cattaert, D and D LeRay (2001) Adaptive motor control in crayfish. Progress in Neurobiology 63: 199-240.
(A one-page excerpt that briefly summarizes the swimmeret motor system).2. Mulloney, B, D Murchison and A Chrachri (1993) Modular organization of pattern-generating circuits in a segmental motor system: the swimmerets of crayfish. Seminars in the Neurosciences 5: 49-57.
(An excerpt giving a basic description of the swimmeret pattern generator).3. Braun, G and B Mulloney (1993) Cholinergic modulation of the swimmeret motor system in crayfish. J. Neurophysiology 70 (6): 2391-8.
(This is the paper to focus on.)4. Mulloney, B (1997) A test of the excitability-gradient hypothesis in the swimmeret system of crayfish. J. Neuroscience 17(5):1860-8.
(An excerpt presenting additional experiments with the cholinergic agonist carbachol.)5. Acevedo, LD, WM Hall, and B Mulloney (1994) Proctolin and excitation of the crayfish swimmeret system. J Comp Neurol 345(4):612-27.
(Abstract only. The paper describes neurons that contain proctolin and that probably activate the swimmeret CPG.)
Read the first and second articles for background on the swimmeret system. Read the third article very closely, paying particular attention to experimental details. Try to imagine exactly how each step of the experiment was done, from dissecting the preparation through data-analysis. This is the article we will discuss most closely in class. Read the excerpt from the fourth article for additional insight about carbachol's action, and the fifth (abstract only) for ideas about where proctolin might act.
Writing assignment
1. Write a three-to-four page paper that you bring with you to lab.
Describe the swimmeret system and discuss the potential sites of action of transmitters that modulate the system. As you read the papers, ask yourself what point each figure is making, and how its component parts contribute to that point. To organize the information about the different drugs and transmitters, try to imagine where on the model of the swimmeret CPG circuit (above) the drugs might act. (No one knows where they actually act.) Base your paper on relevant information from the five readings. It is useful to discuss the readings with your classmates, but you must write your paper by yourself.
There is no need to type out a bibliography for your paper, since all of us have the list of five references. Cite the papers using the author(s) and year in parentheses, as in (Cattaert and LeRay, 2001), (Mulloney et al., 1993), etc. Cite only the most important facts or ideas, not every detail.
Abstracts of
Lab Projects:
(*Smith
campus only)
2. In lab, after discussing the project with your lab partner(s), write a single paragraph that briefly describes an experiment on the swimmeret motor system that will be your group's lab project. Also include a list of drugs that you will need and their approximate concentrations. Put everyone's names on the page. The paragraph's purpose is to describe what you are thinking of doing as a first step in shaping a realistic project. It does not have to be the last word on the subject.
In planning your project, assume that you will employ extracellular electrodes and equipment that you have already used in lab. Use carbachol, proctolin, or another agonist to elicit the swimmeret motor pattern. It is not necessary to give experimental details beyond the overall plan of the experiment. Your experiments do not have to be new. For example, trying to record simultaneously from both sides of a ganglion or both branches of N1 can be an interesting challenge. If you are thinking of a pharmacological experiment, limit yourself to one drug. You can also use a drug (proctolin or carbachol) to initiate the rhythm while you investigate some other aspect (like the timing between ganglia). Keep your experiment simple. You'll want to be able to repeat experiments, and we have only a few weeks.
After the discussion
Try a practice dissection of an isolated nerve cord -- it's worth the time to do it today. See the video and instructions for Labs 9-12.
Links
Supplement: Anatomy of the Crayfish Nervous System.
© 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.