BIO325 Laboratory Guide #4 (2024)
ELECTROPHYSIOLOGY II:
RECORDING FROM THE CRAYFISH IIIs ROOT
falls under category
A
In this lab you will learn and apply the central principles of extracellular recording of nerve action potentials or "spikes" from a peripheral nerve. These are extremely fast (~1-2 msec. duration) and small (<500 V amplitude) signals. The temporal (time) properties of action potentials are similar to those of the pulse-type electric fish, and you should therefore use the PowerLab Scope application with similar recording settings to those used with these fish.
The extremely small signal size makes reduction of environmental electronic noise a crucial consideration. To minimize this noise you will be using every trick at your disposal. The recording area must be surrounded by a grounded Faraday cage, which will serve as an outer shield to pass environmental electromagnetic radiation to ground. All power and recording cords running into the cage, all metal objects in the cage, and the electrodes themselves must also be individually grounded or shielded. Recording at the electrode will be done bipolarly to take advantage of "common-mode" rejection. The signal will be tightly filtered around the frequency of interest, in this case 1 KHz.
In this laboratory, you will be making electrical contact with crayfish peripheral nerves via suction electrodes. The actual live electrode is a bare silver wire, mounted in a hollow plastic tube. This tube, in turn is capped with a small diameter tapered plastic pipette tip. Ringers salt solution is sucked up into this tube, then the surface of the nerve or even a loop of the nerve is sucked up into the tip. If the nerve tightly seals the tip, then a stable electrical contact is maintained between the wire electrode and the nerve itself, via the Ringer's solution. A silver wire spiraled around the outside of the pipette and extending into the salty Ringers solution surrounding the nerve serves as a reference electrode.
You will use a finely-machined micromanipulator to accurately position each electrode, as you practiced in laboratory #1. You must become proficient at using the manipulator while you are looking at the preparation through the dissection microscope. To do this will require both patience and practice. In order to keep the electrode position stable, the entire recording preparation must be mounted on a mechanically isolated platform, consisting of a heavy steel plate suspended on four small inner-tubes.
I. PRELIMINARIES
A. Setup
1) Turn on the PC, if necessary, and open the Scope program. Set it up for recording from input channels 1 and 2. Set each channel for monopolar recording, set the Range to 50 mV, and activate AC and line filters. Set up Scope for repetitive sampling at 100 msec/sweep and the maximum sampling rate (40 kHz).
2) You will be using 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 each 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.
3) 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.
4) Trace all of the connections your recording setup and make sure that you understand what each is for.
B. Noise Check (one person in your group does this, while another does IIA)
1) Start repetitive sampling on Scope. You should both see and hear a fairly high background noise level.
2) Place a Sylgard-lined dish on the microscope stage and fill it to a depth of ~1 cm with cold crayfish Ringers. Position both suction electrode tips in the Ringers, making sure that the spiral reference electrode leads are also immersed. Suck just enough Ringers up into the tip to contact the inner silver wire. The noise level for each channel should drop appreciably as soon as the Ringers makes electrical contact. It is important that you suck Ringers up only into the suction electrode. If you suck Ringers up into the tube and syringe, it will act like a long antennae connected directly to your live recording electrode and produce an unacceptably high noise level.
3) Ideal background noise for each channel should have the following characteristics:
< 20 mV peak-to-peak amplitude (<20 mV on the Scope screen)
broadband noise (similar amplitude at all frequencies)
no obvious standing 60 Hz component to the noise
no slow baseline fluctuations.
If your noise levels do not meet these criteria, check your electrodes, grounding, filter, and amplifier settings. The instructor will help you with this. You will not be able to effectively proceed with neural recording until you have achieved a suitably low background noise level.
4) Before removing the electrodes from the Ringers "bath", be sure to turn the amplifier gain to its lowest setting (x100).
II. Recording and Analysis of Superficial Third Root Spike Activity
In this lab you will follow the general guidance of Crawdad laboratory #2. Review the surgical and recording procedures in the CD guide prior to starting this exercise.
A. Crayfish Preparation (one person in your group does this, while another does IB)
1) Following the Crawdad guide chill a crayfish, cut off the abdomen and pin it out in a sylgard-lined dish, cover with Ringers, place the dish under the dissecting microscope, remove the swimmerettes, and carefully expose the ventral nerve cord in several segments. You can either make individual T- or I-shaped incisions through the cuticle in each segment (as in the Crawdad video).
As an alternative, you can view the video below and follow its procedure. This is the surgical exposure you will ultimately make for recording from muscle and exploring the neuromuscular junction.
http://www.wellesley.edu/Biology/Concepts/Html/crayfishabdomen.html
Regardless of which method you choose, make sure to keep your incisions AS SHALLOW AND SUPERFICIAL AS POSSIBLE and AS CLOSE TO THE MIDLINE AS POSSIBLE. Deep cuts will damage the abdominal nerve cord and lateral cuts will destroy the superficial third roots (IIIs).
2) Examine the abdomen carefully. Only segments with at least one intact IIIs root will be useful for recording. If you have severed the ventral nerve cord anywhere in the abdomen, discard the tail and start over.
3) Position both electrodes over the abdomen.
B. Single Channel Multiunit Recording
1) Suck up a third root. To secure the seal you may try any of three methods: 1) use a pair of fine forceps to crimp the tip of the electrode prior to sucking up the root, 2) carefully place a drop of vaseline against the tip after sucking up the root, or 3) press the electrode tip down against the underlying muscle tissue after sucking up the tip. This third method is the simplest and generally the most practical.
2) There are six distinct motor axons in each third superficial root (IIIs). A good seal with a viable root should show at least three distinct action potential sizes, corresponding to three distinct axons. The signal-to-noise ratio should be at least 10:1 for the largest of these. If you don't have this good a recording, keep trying until you do.
3) Use a plastic probe or small paintbrush to stimulate the hairs on the telson, the hairs on the carapace, and the cut ends of the swimmerette. At least one of these treatments should markedly change the spike activity in IIIs.
Q1: What is the theoretical relationship between axon size and action potential size?
Q2: What is the effect of stimulating the telson hairs? Of bending the telson? Do firing rates in IIIs appear to increase with stimulation? Does stimulation activate any new units (axons)? Which areas of the telson are most effective?
Q3: Ditto for carapace hairs and swimmerette trunks. Do the carapace hairs and/or swimmerette trunks have to be in the same segment as the IIIs nerve root?
4) Tape record at least 5 minutes under the baseline condition and 5 minutes under one condition of continuous stimulation. Make sure that the tape is well-labeled and save it for use in a later lab and/or for later analysis to produce the data sheet items below.
5) Set Scope Sampling to Multiple, then record and save 128 sweeps each at 100msec/sweep under unstimulated and stimulated conditions. Label these records and save them to disk.
6) Save an additional 128 sweeps at a high sampling rate (20 msec X maximum samples/sweep) for use in data sheet item #3 below.
7) You can use either your saved Scope traces or repeated playback of this tape-recorded activity to produce the Data Sheet items below. DO NOT TAKE THE TIME TO PRODUCE THESE PRINTOUTS OR COUNT SPIKES NOW. Proceed directly to the next section below.
C. Dual-Channel Multiunit Recording
1) Using both suction electrodes, suck up two IIIs roots - either both roots in the same segment (contralateral), or two roots on the same side of adjacent segments (ipsilateral).
2) Make sure that each channel show at least three distinct AP sizes with a signal-to-noise ratio of at least 10:1 for the largest unit. If you don't have this good a recording, keep trying until you do.
3) Again, use a plastic probe of brush to stimulate the hairs on the telson, the hairs on the carapace, and the cut ends of the swimmerettes and note the effects of each.
4) Tape record at least 5 minutes of two-channel activity under the baseline condition and 5 minutes under one condition of continuous stimulation. Make sure that the tape is well-labeled and save it for use in a later lab.
5) Set Scope Sampling to Multiple, then record and save 128 sweeps each at 100 msec/sweep each under unstimulated and stimulated conditions. Label these records and save them to disk. Again, you can use either live recording to PowerLab, or repeated playback of tape-recorded activity to produce the Data Sheet items below.
6) You can use either your saved Scope traces or repeated playback of this tape-recorded activity to produce the Data Sheet items below. DO NOT TAKE THE TIME TO PRODUCE THESE PRINTOUTS OR COUNT SPIKES NOW. Proceed directly to the next section below.
D. Independent Project
Design a simple test addressing an experimental question of your own design which centers on the recording techniques of this lab. As examples, you might test the effects of the ionic makeup of the Ringers bath, the effects of cutting the nerve cord proximal or distal to the recording site, the effects of cutting the IIIs root distal to the recording site, or the effects of bath temperature.
Data Sheet Item #7:
Document your independent project design, experimental question, and results with appropriately labeled and annotated printouts, tables/ and/or graphs.
IV. CLEANUP AND SHUT DOWN
1) Exit Scope, turn off the PowerPC, and turn off all amplifiers and lights.
2) Dispose of all large crayfish parts in plastic bags in the freezer in room 104. Dispose of all small crayfish parts into the trash. Clean and rinse out your sylgard-lined recording dish and all the surgical instruments which you used.
3) Flush distilled water and air through your suction electrodes.
4) Clean up any water, Ringers, or dried salt anywhere in your recording station.
5) Make sure that the rest of the room, especially the four general-use student tables are all completely clear.
V. PREPARATION OF THE LAB DATA SHEET
Your data sheet should include at least FIVE 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