BIO325 Laboratory Guide #20 (2024)
MOTOR CONTROL SYSTEMS:
SPONTANEOUS ACTIVITY AND EVOKED POTENTIALS
IN THE CRAYFISH ROOT II
falls under category
A
In previous labs you have studied action potentials, muscle resting potentials, and neuromuscular junctions in the superficial third nerve root/superficial flexor muscle system of the crayfish. In this lab you will continue to explore the organization of the crayfish sensorimotor systems by looking at both spontaneous activity and electrically evoked potentials in the second nerve root. This nerve root innervates primarily the extensor muscles of the abdomen. It also carries sensory axons from the tactile hairs and the stretch receptors which you studied in lab 21. It is also the easiest nerve root to record from due to it size and position.
As in previous labs, you will use the isolated crayfish abdomen, maintained in Ringer's solution. A slight variant of the surgical procedure (see below), will be used to expose the entire abdominal nerve cord and all of the nerve roots. Spontaneous, background activity in a resting tail will reflect primarily action potentials in the efferent motor axons. Afferent sensory axons should be quiet, because in this surgical preparation these axons will be cut between the sensors and the recording site.
Electrically stimulating the abdominal nerve cord proximal to the root II with a sufficiently large and short square-wave pulse will result in an "evoked potential" (EP) response, which is, to some degree, "time-locked" to each stimulus pulse. This evoked potential response will have two components. The earlier component is a "compound action potential" (CAP) that results from directly driving each of the afferent sensory fibers "antidromically", that is backwards down the axon from the nerve cord and out into root II. This component may vary in amplitude as stimulus intensity is increased and more axons are stimulated, but should have a very fixed time course relative to the stimulus pulse. Because no synapses are involved, this component should also persist and remain unchanged as stimulus frequency in increased, provided the inter-pulse interval remains longer than the refractory periods for the axons.
The later component of the evoked response will consist of superimposed individual action potentials from motor axons running in root II. Each of these axons will be separated from the stimulation site by one or more synapses. This component will be delayed due to the synaptic delays. It will also be much more variable, both in time course and in amplitude. Finally, this component should be significantly diminished by high frequency stimulation, as individual synapses are inactivated and their postsynaptic responses disappear.
This lab is based on the following laboratory exercise developed by Richard Olivo at Smith College. This link will provide additional background information, as well as a video link detailing the surgical and recording procedure.
http://www.science.smith.edu/departments/NeuroSci/courses/bio330/labs/L7cns.html
I. PRELIMINARIES
A. Crayfish Surgery
One person in your group does this, while the other does IB.
1) Chill a crayfish, cut off the abdomen as close as possible to the thorax, and pin it out in a Sylgard-lined bowl. Immerse the abdomen in crayfish Ringer's.
2) Following the procedures detailed in the Olivo vide link above, use your fine iris scissors to make two shallow cuts at the lateral margins of the ventral cuticle. Extend each cut the length of the abdomen. Peel back the just the ventral cuticle towards the telson, being careful to leave the nerve cord and nerve roots intact within the abdomen. You may have to do some further careful dissection of the filmy "under-cuticle" to fully expose the nerve cord and roots. When you are finished, you should have completely exposed the intact ventral nerve cord. Each of the more posterior segments should have long first and second nerve roots cut only at their lateral extremities. Try to actually touch the nerve cord and roots as little as possible with the metal instruments.
B. Recording Setup
1) Turn on the PC and the PowerLab box, if necessary, and open the Scope program. Set it up for recording from input channel 1 only. Set this channel for monopolar recording, set the Range to 50 mV, and activate AC filter. Set up Scope for repetitive sampling at 100 msec/sweep and the maximum sampling rate.
2) Under the Setup menu of Scope select Stimulator . . ., then set the stimulator Mode: to Pulse, Delay to 5msec, Duration to 1msec and Amplitude to about 4 Volts. Under the Display menu select Display Settings . . . , then set the Graticule to a grid pattern and Channel A to an attractive color. Under the Display menu choose Overlay Stimulator and place the stimulator trace at the top of the window.
3) Connect the Output + of the PowerLab box to the TRIGGER IN of the electronic stimulator with a BNC cable. Connect the output cable to the stimulator and to the stimulation probe mounted on one of the micromanipulators. The electronic stimulator must remain TURNED OFF at this point.
4) You will be using one channel of the Model 1800 two-channel AC amplifier. Make sure that the amplifier ground is connected to the common cage/PowerLab ground. Turn on the amplifier. Set the live amplifier channel to Rec and x1000 gain. Turn on the 60Hz notch filter and set the Low- and High-cutoff filters to 100 Hz and 5 kHz, respectively. Make sure that Scope channel A is recording from this live channel of the amplifier.
5) Check to make sure that the suction electrode corresponds to the correct channel of the model 1800. Blow any residual water/saline out of the suction electrode tip. Make sure that all three preamplifier leads are correctly connected to the suction electrode.
6) Turn on the audio amplifier. Make sure that the Mono switch is out and the selector is set to Tuner (this corresponds to directly monitoring the output of the 1800 amplifier to the PowerLab inputs). Make sure that the audio selector switch is set to STR.
7) Position the suction electrode tip in the bath and suck up just enough Ringer's solution to fill the suction tip. Start the repetitive scope sweep and verify a normal and acceptable background noise level on both the screen and the audio monitor.
II. RECORDING AND ANALYSIS OF ROOT II ACTIVITY
A) Background Activity
1) Position the crayfish abdomen so that the telson is pointed directly towards the back of the Faraday cage and the cut end of the abdomen is pointed towards you. Carefully examine the crayfish abdomen. Each ganglion should have four large nerve roots extending directly out of it. The more caudal pair are the first roots and the more distal pair are your targets the second roots. Just caudal to the ganglia two more pairs of nerve roots emerge - the larger deep third roots and the tiny superficial roots. If you have ANY questions about the anatomy just discussed, ask the instructor for clarification before proceeding. If you have severed the ventral nerve cord anywhere along its path through the abdomen, you will have to discard the crayfish abdomen and start over with a new one.
2) Lift the rostral cut end of the nerve cord using a small glass hook. For ONLY THE FIRST TWO SEGMENTS (the ones closest to the thorax), cut all four roots on each side close to their origins in the ventral cord. Gently free up the cord from below, so that the cord in these first two abdominal segments can be lifted clear of the underlying muscle tissue. You will use your fine iris scissors to cut each nerve root, but manipulate the roots and nerve cord using only the glass hooks. Try not to touch the nerve cord or roots with any metal instruments.
3) Choose a long second nerve root on one of the posterior ganglia. Position your suction electrode and suck up this second root, as described in the Olivo video clip.
4) Start Scope and monitor the ongoing background activity. You should see spontaneous activity from multiple motor units (there are actually thirteen in each II root). You may be able to increase this background activity level by stimulating the hairs on the telson with a brush - to reduce background noise be sure to ground yourself to the cage before reaching in.
5) It will probably be most convenient to save samples of your background activity by setting Sampling to Multiple. Stop the Scope sweeps when you are finished.
B) Response to Stimulation
1) Position the stimulating probe so that the silver hooks are close to the cut end of the nerve cord. Using only the glass hooks, gently lift the nerve cord onto the silver stimulating hooks. Raise the stimulating probe vertically to lift the stimulating hooks and the cut end of the nerve cord completely out of the Ringer's bath. You may have to lower the level of the bath Ringer's to accomplish this.
2) Set the electronic stimulator to deliver a single single monopolar pulse. Set the delay to the minimum possible setting, the duration to 0.1 msec, and the amplitude to 0.1 volts. When you are sure that your settings are correct, turn the electronic stimulator On.
3) Set up Scope for Repetitive sweeps, with a 1 second interval between sweeps. Change the sweep duration to 50 msec at maximal sampling rate.
4) Start Scope. Verify via the electronic stimulator monitor LED that stimuli are in fact being produced. Each Scope sweep should show a transient stimulus artifact at exactly 5 msec into the sweep.
5) Increase stimulus intensity and/or stimulus duration and look for a longer evoked potential response to the stimulation. Do not go beyond 5 volt stimulus intensity or 1 msec duration.
6) A indicated in the introductory section above, the evoked response should have a fairly constant initial component - the sensory axon CAP, and a much more variable later component - the superimposed APs of motor axons.
7) Once you have established a reliable evoked potential response, stop the Scope sweeps, clear the Scope screen, set Sampling to Multiple and 32 samples, then Start the Scope sweep.
C) Stimulation Frequency Effects
1) Save your data from the previous section and clear out the traces.
2) Under the pull-down Sampling menu, eliminate the 1 second interval between successive samples.
3) Start Scope to collect 32 more samples at the new, higher stimulation rate.
D) Stimulation Intensity Effects
1) Save your data from the previous section and clear out the traces.
2) Set Sampling back to Repetitive and restore the 1 second interval between samples (and stimuli).
3) Reduce the stimulus amplitude to near absolute threshold for the evoked response.
4) Set Sampling back to Multiple and collect 32 sweeps at this setting.
5) Save this data, then clear the data from Scope.
6) Double the stimulus amplitude, then repeat steps 4 and 5. Do not go above 5 volts amplitude.
7) Repeat step 6 twice more (if possible - but stay below 5 volts amplitude!), for a total of four sets of traces.
E) Effects of Nicotine on Second Root Activity
1) Save your data from the previous section and clear out the traces.
2) Turn off the stimulator.
3) Eliminate the 1 second delay between successive traces. Change the sweep duration back to 100 msec at the highest possible sampling rate.
4) Save 32 traces of background activity.
5) Apply ~5 drops of nicotine solution directly over the ganglion from which you are recording.
6) Immediately save 32 more traces of background activity.
7) After 1 minute save 32 more traces.
8) After 4 more minutes save 32 more traces.
F) Shutting Down
1) When you are sure that you have saved all of the data that you need, quit Scope, turn off the amplifier, and turn off the PowerLab box.
2) Retract the suction electrode and flush it out with air.
3) Retract the stimulating probe and flush it of with water.
4) Properly dispose of your crayfish tail.
5) Properly clean all of your instruments.
6) Give Henaa an appreciative pat on the head for all of the work that she did in testing out and preparing this lab.
III. PREPARATION OF THE LAB DATA SHEET
Your data sheet should include THREE of the items described in the boxes above. Make sure that the axes of all of the graphs and print-outs are labeled and calibrated. You should certainly discuss your results and the answers to the questions with your partners and others in the lab. However, please work independently when you prepare your data sheet.
The writeup
for this lab
falls under category
A