BIO325 Laboratory Guide #15 (2024)
SYNAPSES II:
POSTSYNAPTIC POTENTIALS IN THE CRAYFISH
NEUROMUSCULAR JUNCTION
falls under category
A
This laboratory exercise and exercise #19 are the final two labs involving the crayfish third superficial nerve root, the associated superficial flexor muscles, and the neuromuscular junctions between them. In this lab you will combine the techniques you developed in previous exercises to simultaneously record neuronal action potentials extracellularly from the third superficial root and muscle resting and postsynaptic potentials (RPs and PSPs) intracellularly from single muscle cells. Prior to starting this lab you should review Crawdad labs 2 and 4, as well as the associated laboratory guides 5 and 7 from this course. This exercise will follow Crawdad lab #8, so please preview the techniques on the video clip.
As you work through this lab you will explore the properties of muscle postsynaptic potentials, including the range of durations and amplitudes, and the summation which occurs when PSPs overlap in time. You will also investigate the relationships between muscle PSPs and the presynaptic neuronal action potentials which elicit them. Remember that in invertebrates an individual muscle cell may receive synapses from several axons, as well as multiple synapses from a single axon (see Crawdad Appendix A). This means that each muscle cell may show up to six distinct classes of PSPs, corresponding to the six motor axons which run in the IIIs root. Each class of PSPs will have a characteristic shape and amplitude, as well as an obligatory temporal relationship with one of the six classes of presynaptic action potentials. By recording from several muscle cells you can also produce a rough map of the spatial pattern on innervation of these six axons onto the superficial flexor muscle.
I. RECORDING PREPARATIONS
A. Preparing Microelectrodes
1) Pull, fill, and test at least 4 microelectrodes. Electrode impedances of 2-5 MW are ideal, but impedances in the 1-2 MW 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 #8. 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. Set it up for recording from input channels 1 and 2. Channel A will monitor the extracellular suction electrode. Set it for monopolar recording, set the Range to 50 mV, and activate the AC filter. Channel B will monitor the intracellular microelectrode. Set it 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 ) Turn on the Model 1800 two-channel AC amplifier. Make sure that the amplifier ground is connected to the common cage/PowerLab ground. Set the left-hand amplifier channel to Rec and x1000 gain. Turn on the 60 Hz notch filter and set the Low- and High-cutoff filters to 100 Hz and 5 kHz, respectively.
5) 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 1600 amplifier to the PowerLab inputs). The left audio channel will monitor the extracellular AP recording and the Right audio channel will monitor the intracellular PSP recording.
6) Lower the suction electrode into the bath and perform a noise check to make sure that the extracellular recording noise is <+20 mV.
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. RECORDING NEURONAL ACTION POTENTIALS AND
MUSCLE POSTSYNAPTIC POTENTIALS
A. Recording APs and EPSPs
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 #8, 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. Position the suction electrode, advance it, and suck up the nerve root. Make sure that you have at least three different AP amplitudes represented in the recording from this electrode.
3) Mount a micropipette microelectrode in a half-cell electrode hoder, and mount this into the Neuroprobe headstage. Position the microelectrode, advance it, and penetrate a muscle cell. It is best to penetrate the cell near its center. PSPs will appear as small (1-10 mV) transient (2-5msec) deflections away from the resting potential value.
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) Switch to Multiple sampling to save traces.
6) Save enough traces to produce the following three data sheet items. It might also be a good idea to record at least five minutes of this AP activity to audio tape.
7) Use a small brush to stimulate the hairs on the telson and record the resulting APs and PSPs. Remember to ground yourself by wetting the fingers on your free hand and lightly touching the cage, in order to reduce recording noise when you reach into the cage.
B. Mapping Innervation of the Superficial Flexor Muscle
1) Record from several muscle cells, moving systematically along a line from near the medial edge of the muscle to near its lateral edge. Keep the electrode positioned near the middle of each muscle cell (on a rostro-caudal axis). Again, it would be a good idea to same a sample of each of these recordings to tape.
2) For each muscle cell, record under both unstimulated and stimulated conditions.
III. MINI-INDEPENDENT STUDY
Design and conduct an independent study of your own design, which explores some aspect of the relationship between IIIs action potentials and superficial flexor muscle PSPs. Possible topics might be:
the effects of changing the muscle resting potential(s) via ionic substitutions in the bath
the effects of changing the muscle resting potential by injecting current into the muscle cell
the effects of neurotransmitter antagonists or agonists
Additional ideas may be found at the end of Crawdad lab #8. Be sure to keep good records of your procedures and label your result traces as you go.
IV. SHUTTING DOWN
Perform the following steps when you are finished:
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 both amplifiers.
3) Properly discard all microelectrodes.
4) Flush out the half-cell electrode holders 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 103 and store all crayfish parts in the freezer.
V. 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
falls under category
A