Biological Sciences 330, Smith College | Research in Cellular NeurophysiologySwimmeret Central Pattern GeneratorRevised: February 19, 2019 |
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Experimental methods.View the video: Central Pattern Generator for Crayfish Swimmerets, which shows the procedure for isolating a crayfish nerve cord and recording from the first roots. Recording from an isolated crayfish nerve cord with pin electrodes enables us to detect the patterned activity produced in the first roots during "fictive locomotion" of the swimmerets generated by central pattern generator circuits in the ganglia. The swimmeret muscles receive axons from motoneurons 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 electrode method described below. |
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To prepare an isolated nerve cord, follow the procedures outlined in an earlier lab 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 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 the ganglia 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. |
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Monitor neural activity on your oscilloscope screen, your audio monitor, your computer (using PowerLab), and directly on your chart recorder. Rhythmic CPG bursts will be easy to hear, and you will see the bursting pattern easily on the computer screen (at slow scrolling speeds) or on your chart recorder. PowerLab will be the most versatile, since you can later zoom in on details of the activity, integrate the records or analyze spike rates, and create materials for your presentation.
Some helpful hints:
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Supplement: Anatomy of the Crayfish Nervous System. Background information about the crayfish swimmeret system. Appendix: Capturing Oscilloscope Screenshots Appendix: Using EasyGraf Chart Recorders. |
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