BIO325 Laboratory Guide #17 (2024)
SYNAPSES IV:
NEUROPLASTICITY IN THE CRAYFISH
NEUROMUSCULAR JUNCTION
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This laboratory exercise is a follow-up to laboratory 17, in that it involves the crayfish third superficial nerve root, the associated superficial flexor muscles, and the neuromuscular junctions between them. In this lab you will again surgically expose the third superficial root and associated superficial flexor muscles. You will again attach a suction electrode to the nerve root and record intracellulary from individual muscle cells. However, in this lab you will electrically stimulate the nerve root to directly produce muscle PSPs. In this way it is somewhat analogous to the plant action potential lab. Furthermore, you will be testing for specific phenomena involving changes in PSP amplitude over time in response to repetitive nerve stimulation. Prior to starting this lab you should review Crawdad labs 2, 4, and 8, as well as the associated laboratory guides 5, 7, and 17 from this course. This exercise will follow Crawdad lab #9, so please preview the techniques on the video clip.
I. RECORDING PREPARATIONS
A. Preparing Microelectrodes
1) Pull, fill, and test at least 4 microelectrodes. Electrode impedances of 2-5MW are ideal, but impedances in the 1-2MW range are useable.
2) Spare electrodes may be imbedded in clay in a petri dish container for storage up to several hours, but are not useable after a day or more.
B. Crayfish Surgery
1) Review the CD video guide for Crawdad Laboratory #9. See also Appendix A for explanatory figures and a background on the crayfish superficial abdominal flexor system.
2) Isolate and pin out a crayfish tail in a Sygard-lined dish, then immerse it in cold crayfish Ringers. Choose at least two segments. Make a midline incision trough the cuticle in each segment to expose the nerve roots. Carefully expose the superficial flexor muscles on both sides of each segment. Important things to watch out for in this surgery are:
- use a fresh scalpel blade and keep it flat - DON'T CUT TOO DEEPLY
- don't damage the muscle insertion line which runs medio-laterally
- make sure that IIIs (the third superficial root) is intact and undamaged
If any of these conditions are not met, then that half-segment cannot be used.
3) Don't proceed to the recording phase until you have at least four intact half-segment muscle exposures.
C. Recording Setup
1) Turn on the power to the Model 1600 Neuroprobe Amplifier and allow it to warm up for 5 minutes. Set all of the switches and knobs for standard intracellular recording.
2) Turn on the microscope and fiber-optic lights. Fill an extra Sylgard-lined recording dish with about 1 cm of cold crayfish Ringers. Place the reference/ground pellet in the bath.
3) Turn on the PC, if necessary, and open the Scope program. Under Display:Computed Functions choose ChA Only. Under Display:Overlay Stimulator select this option. Set ChA for monopolar recording, set the Range to 200 mV, but do not activate any filters. Set up Scope for repetitive sampling at 5 msec/sweep and the maximum sampling rate (40 kHz).
4) 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. You will be using the PowerLab stimulator only to trigger the electronic stimulator. The output (most importantly the timing) of this stimulator will appear as a trace across the top of each Scope sweep. 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.
5) 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 attach long alligator clip test leads to each banana plug. Connect the positive (non-tab) alligator clip to the live lead of the suction electrode. Connect the negative (tab) alligator clip to the reference lead of the suction electrode. Using another long alligator clip connect the black shield lead of the suction electrode to the cage ground.
6) Connect the positive output of the stimulator to the PowerLab CH1+ input and the negative output of the stimulator to the PowerLab CH1- input, using BNC to double-banana cables.
7) Mount a filled microelectrode in a half-cell holder and plug the holder into the Neuroprobe headstage. Lower the microelectrode into the bath. Adjust the DC OFFSET switch and knob until the meter reads 000. Retest the electrode and adjust the Cap Comp. to square up the calibration signal.
8) Check all for the recording connections and make sure that you know what each one is for. Now is the time to ask questions.
II. STIMULATING THE SUPERFICIAL THIRD ROOT AND
RECORDING EVOKED MUSCLE POSTSYNAPTIC POTENTIALS
In this laboratory exercise you will explore another aspect of the crayfish neuromuscular junction, namely the plasticity or changeability of the synapses. After demonstrating that you can elicit PSPs in individual muscle cells by directly stimulating the third superficial root you will look for evidence for two neuroplasticity phenomena. The first of these is post-synaptic (two-pulse) facilitation. The PSP response will tend to increase in amplitude with each successive stimulus pulse, if successive stimuli are presented closely enough together. The second phenomenon is called post-tetanic potentiation and has a longer time course. If a "tetanizing" train of closely spaced stimuli are delivered across a synapse, the synapse will be strengthened for some time afterwards. Again, the methodology for these experiments will closely follow Crawdad lab exercise #9.
A. Evoking PSPs
1) Retract the electrodes from the bath in the empty dish, then replace this with your prepared crayfish abdomen.
2) As indicated in Crawdad lab #9, it is best to establish a stable contact between the suction electrode and the nerve before you start penetrating muscle cells. Choose a hemi-segment with an intact IIIs root and undamaged superficial flexor muscle. Cut the superficial third root near the ganglion. Position the suction electrode, advance it, and suck up the cut end of the nerve root. To create a tight seal you may either apply a small dab of vaseline to the end of the suction electrode, or advance the tip against the underlying muscle as you suck in the nerve root.
3) Position the microelectrode, advance it, and penetrate a muscle cell. It is best to penetrate the cell near its center. Because the nerve root has been cut and is not carrying spontaneous APs, you should not see spontaneous PSPs in the muscle. If you have an extremely low noise recording you may, however see spontaneous miniature end-plate potentials (MEPPs) associated with random presynaptic fusion and release of individual neurotransmitter vesicles.
4) Once you have established that you are inside a muscle cell with a healthy resting potential of at least -40 mV, change the PowerLab CH B filter setting to AC and range to 10 mV. Slow the time scale to 50-100 msec (at the maximum sampling rate). This will highlight the PSPs.
5) Set the electronic stimulator to produce single 1V x 10 msec pulses with a minimal delay. Make sure that the stimulator polarity is set to negative. (You are stimulating the outside of the nerve fibers so a negative pulse is depolarizing.) Turn on the electronic stimulator.
6) Trigger single Scope sweeps using the START button. If each sweep produces one or more PSPs, reduce the amplitude and/or the duration of the stimulus pulse until only a single EPSP is elicited with each sweep. Continue to reduce the stimulus amplitude/duration to a near-threshold setting. If your initial stimulus settings are insufficient to produce EPSPs, gradually increase the intensity of the stimulus pulse. If you have to go above 10 V, then try another muscle cell.
B. Paired-Pulse Facilitation
The idea here is to first demonstrate, then quantify the facilitation in EPSP amplitude produced by successive stimuli.
1) Establish settings which reliably produce a single clear EPSP in response to nerve stimulation.
2) Set the PowerLab stimulator mode to Multiple, set the number of stimulus pulses to 10 and the interstimulus interval to 150ms. Set the Scope time base to a 1 second sweep at the maximal sampling rate.
3) Trigger a single PowerLab sweep. You should see a train of PSPs in response to the train of stimulus pulses being delivered. Furthermore, each PSP should be progressively larger in amplitude.
4) Reduce the number of stimulus pulses to 2, with a 1000ms (1 second) interval between them. As outlined in Crawdad lab #9, quantify the time-dependence of facilitation by gradually reducing this interstimulus interval. For each interstimulus interval you can compute a facilitation index as the ration of the height of the second PSP to that of the first (h2/h1). Video 9.4 and the accompanying figure in the Crawdad manual show you how to measure h2 when the two PSPs start to overlap in time.
C. Post-Tetanic Potentiation
For this part of the experiment the methodology is a little complex. What you want to do is trigger and record a single PSP, then deliver a prolonged train of closely-spaced stimuli which drive the synapse at a relatively high frequency, then after a suitable interval trigger and record another single PSP. The intervening tetanizing train of stimuli should increase the amplitude of the second PSP relative to the first. Here is the sequence of steps to accomplish this. You will need a stopwatch for the timing.
1) Reset the PowerLab stimulator to the single pulse mode. Check to make sure that your trigger pulse is still 1msec x 4 volts with a 5msec delay.
2) Reset the Scope time base to a 50 msec sweep at the maximal sampling rate.
3) Set the electronic stimulator FREQUENCY knob to 20 Hz.
4) Trigger a single Scope sweep and verify that a single EPSP has been produced and recorded. This is your "pre-tetanic" EPSP.
5) Click the MODE switch on electronic stimulator up to the REPEAT position, hold it there for 5 seconds, then click it back to the OFF position. You have just delivered a "tetanic" train of stimuli to the nerve and muscle.
6) After 30 seconds trigger another single Scope sweep. This is your "post-tetanic" EPSP.
7) Save and/or label this pair of Scope sweeps.
8) Repeat steps 4-7 with successively shorter post-tetanic delays (step 6), down to 1 second. Wait at least 5 minutes between successive trials.
9) Calculate facilitation index (hposttrain/hpretrain)values for each post/pre pair of EPSPs.
10) Set up Scope to produce 64 multiple sweeps with a 100 msec time base, set to maximal samping rate. Turn off the PowerLab stimulator. Initiate sampling on Scope, then immediately click the electronic stimulator MODE switch to REPEAT, hold it there for 5 seconds, then click it back to OFF.
11) Examine the set of 64 continuous stimulation Scope traces to determine what happened to PSP amplitude during the tetanizing train of stimuli.
III. SHUTTING DOWN
Complete the following steps before leaving the lab:
1) Make sure that you have saved all of your data to the hard drive, then quit Scope. Turn off the PowerLab box.
2) Turn off the amplifier and the stimulator.
3) Properly discard all microelectrodes.
4) Flush out the half-cell electrode holder and the suction electrode with distilled water, then air and store them dry. Flush out your microfil fiber needle with distilled water, then air.
5) Make sure that both the microscope and fiber-optic lights are turned off.
6) Make sure that both micromanipulators are magnetically secured to the steel plate.
7) Make sure that the Microelectrode R/C Meter is turned off.
8) Return all solutions to the refrigerator in 104 and store all crayfish parts in the freezer.
IV. PREPARATION OF THE LAB DATA SHEET
Your data sheet should include at least FOUR 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
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