The web browser you are using does not have features required to display Crawdad correctly.
Please use the most recent version of one of the following:
If you have not already done so, read Appendix A, Crayfish Neuromuscular Preparation, for background. This lab exercise is the first in the sequence introduced there. You will record from the superficial branch of abdominal nerve 3, which controls the superficial flexor (SF) muscle of the crayfish abdomen (tail).
Since the superficial branch of nerve 3 has a small number of axons, individual motor neurons can be discriminated by the amplitudes of their extracellularly recorded action potentials. The amplitude of an extracellular action potential is a function of the diameter of the axon in which it travels. Nerve 3 has a wide range of axon diameters, so most of the axons produce action potentials of different amplitudes. You will use this fact to determine the number of axons in nerve 3 based on your extracellular recording data. In Lab 3, Functional Morphology, you may test your hypothesis with a staining technique that reveals the number, position, and morphology in the central nervous system of the motor neurons from which you record in this lab.
Before starting, look at the methylene blue-stained specimen that was prepared in advance (it will resemble Figure A.2). This specimen shows nerve 3 leaving the ganglion, projecting to the superficial flexor muscle, and branching out over the surface of the muscle. At high magnification, you should be able to see axons in nerve 3. Look closely at the nerve as it enters the muscle. Use this specimen to get a feel for the location and depth of the nerve below the cuticle.
If this is your first experience with live dissection, read Appendix B, Dissection Tips, for suggestions that will make the dissection go more smoothly.
The ventral nerve cord and two ganglia (under the sternites) should now be visible. Note the two thick nerves directly leaving the more anterior ganglion. The first of these (nerve 1) contains axons of motor neurons innervating the swimmerets as well as sensory receptor axons going back to the ganglion. The second one (nerve 2) contains axons of motor neurons innervating the extensor muscles as well as sensory receptor axons going back to the ganglion. Nerve 3 exits the ganglion posteriorly to the other two nerves and may seem to come out of the ventral nerve cord itself. It is thinner than the first two nerves and may be difficult to see at first. This superficial branch of nerve 3 contains only motor axons innervating the superficial flexor muscle (Kennedy and Takeda, 1965b). A deep branch of nerve 3 contains motor neurons innervating the deep flexor muscles (Kennedy and Takeda, 1965a).
Figure 2.1 shows the setup for extracellular recording; Video 2.3, Nerve 3 Recording, shows the entire recording sequence. Before starting to record from the nerve, empty the saline out of the dissecting dish and replace it with fresh cold saline. You should replace the saline with fresh cold saline every half hour or so, but there’s no need to interrupt a good recording to do this.
Bring the suction electrode into the saline and pull gently on the syringe until the tip of the electrode holds enough saline to reach the wire inside the electrode. Place the electrode next to nerve 3 and gently suck a loop of the nerve into the electrode tip. Unless there is a lot of electrical noise, spontaneously firing action potentials should be obvious on the oscilloscope and speaker. Set the oscilloscope to a fast time base (0.5 to 2.0 ms/div). Note the shape of an action potential. Can you explain why it has this shape (see Lab 1, Membrane Properties)?
See Appendix C, Recording Tips, for general information about recording and troubleshooting procedures.
Observe the spontaneous activity of the axons in nerve 3. Note that the action potentials are of a few consistent different sizes, representing individual all-or-none action potentials, and that some sizes occur more often than others. Measure and record the sizes of the action potentials and their frequency of occurrence. Make a histogram of number of action potentials vs. amplitude. On the basis of this histogram, make a hypothesis about the number of motor axons in nerve 3.
Different axons in nerve 3 have different patterns of activity, which can be changed by stimulating reflexes that activate the superficial flexors. One such circuit involves the tail fan. Using a nonmetallic object, stroke hairs along the edge of the tail fan or push gently on the tail fan. Because the ear is better at frequency analysis than the eyes, changes in activity may be more evident in the audio monitor than on the oscilloscope. Note changes in the amount of activity. Do any new sizes of action potential appear during stimulation? How long does the altered motor pattern last after the stimulus? Now try gently moving the swimmeret stumps back and forth in the segment from which you are recording and in adjacent segments. How does this affect neural activity? Make two more histograms of number of action potentials vs. amplitude, one with data collected after stimulating the tail fan and one after moving the swimmeret stumps. Compare these to before-stimulation histograms.
When collecting data for these stimuli, it is important to compare stimulated responses with unstimulated activity occurring immediately beforehand. It is also important to compare the same duration of recording. For example, record 10 s of baseline activity, then 10 s of telson stimulation. After activity returns to normal, record a new 10 s of baseline followed by 10 s of swimmeret stimulation. Compare each stimulated response with the baseline section immediately before. That way, if the electrode shifts and the recording amplitude changes over a long period of time, your comparisons will still make sense.
If time permits, you can follow up on questions raised by the above experiments. For example, how does the reflex response vary with stimulus location? You could map this by systematically stimulating different parts of the tail. How does reflex activity change with repetition of the stimulus? How long does the reflex activity outlast the stimulus? How are spontaneous and reflex activity altered by neuromodulators?
Clean up any spilled saline and rinse the ground electrode with distilled water. Expel saline from the suction electrode and rinse it with distilled water. Put the crayfish tail in the freezer along with the frozen heads and rinse the dissecting dish with fresh water.