Biological Sciences 300/301, Smith College | Neurophysiology

Checklist for Lab 7: Motor Units in the Crayfish Nerve Cord

http://www.science.smith.edu/departments/NeuroSci/courses/bio330/labs/labcheck7.html

UPDATED: March 11, 2015

Knowing of my growing curiosity about invertebrate animals, particularly about the crayfish, [Harry] Grundfest suggested that I set up an electrophysiological recording system with Crain's help. I could use the system to replicate Hodgkin and Huxley's experiment by recording from the large axon of the crayfish, which controls the animal's tail and thus its escape from predators. This crayfish axon is smaller than that of the squid but nonetheless very large.

Crain showed me how to manufacture glass microelectrodes for insertion into individual axons and how to obtain and interpret electrical recordings from them. It was in the course of those experiments -- which were almost laboratory exercises, since I was not exploring new ground scientifically or conceptually -- that I first began to feel the excitement of working on my own. I connected the output from the amplifier I was using to record the electrical signal to a loudspeaker, as Adrian had done thirty years earlier. Whenever I penetrated a cell, I, too, could hear the crack of an action potential. I am not fond of the sound of gunshots, but I found the bang! bang! bang! of action potentials intoxicating. The idea that I had successfully impaled an axon and was actually listening in on the brain of the crayfish as it conveyed messages seemed marvelously intimate. I was becoming a true psychoanalyst: I was listening to the deep, hidden thoughts of my crayfish!

Eric Kandel, In Search of Memory (NY: WW Norton & Company, 2006) pp 108-109.

Background

1. Prior to lab, from the Web instructions for Lab 7, review:

  • Part 1, the functional anatomy of the crayfish nerve cord, including the roles of the three "roots" (nerves) supplying the swimmerets (1st root), the extensor muscles (2nd root), and the flexor muscles (3rd root). You may also wish to preview the Web-based supplement on the Anatomy of the Crayfish Nervous System. You and your lab partner(s) will work with digitized serial sections of a ganglion during lab.
     
  • Parts 3, 4 and 5, the equipment, dissection, and experimental procedures. View the video Motor Units in Crayfish Abdominal Ganglia to see the procedures.

Neuroanatomy

2. In lab, working with the supplement on the Anatomy of the Crayfish Nervous System, first look at images of backfilled motoneurons from the first root. Then work with all three sets of serial sections through an abdominal ganglion, looking for the landmarks listed in the online instructions. Capture a computer screenshot of a serial section that shows details of a first or second root leaving the ganglion.

Equipment

3. Set up the chart recorder for direct recording. Also set up connections to the PowerLab input unit.

Dissection

4. Dissect the abdominal nerve cord and free several roots. Place a suction electrode for recording from the cut roots. Try several roots to see which gives you the best activity.

Experimental
Procedure

5. (a) Capture records of spontaneous neural activity in a root using three different methods:

  • Trigger the sweep on a large spike and capture screenshots of that spike and other spikes that appear in the same sweep. Also observe activity at a slow sweep speed. Stop the sweep, and expand the horizontal scale while moving the horizontal position to inspect different sections of the captured data. Make one or more additional screenshots.
  • Plot some of the spontaneous activity directly on your chart recorder. Try different chart speeds to examine the resolution and level of detail.
  • Use your PowerLab and Chart software to capture spontaneous firing as a file. (See the appendix: Capturing Data with PowerLab and Chart.) On your computer screen, expand Chart's timescale and scroll through the captured record to visualize individual spikes. Use the Spike Histogram module to isolate and plot the firing of an individual axon's spikes.

(b) Using the chart record, identify several of the largest units (spikes in different motor axons) and put numbers above them to mark when each unit fires. Calculate each unit's average firing rate during a 10 to 20-second sample.

[(b2) Although an optional procedure is described to electrically stimulate the connective, we will not do that procedure. You can see the experiment and some typical results in the lab video.]

(c) Prepare your computer Chart software and also the EasyGraf chart recorder to capture the response to nicotine as both a computer file and a paper chart record. After recording some baseline activity as a control, put a drop or two of nicotine on the ganglion nearest the electrode and record the response.

Posting
data

6. Create a single neat display for the bulletin board that summarizes your group's observations. Make sure that you label each record explicitly, and that your names are on everything you post.

  • On a Pages or Word page, arrange sample oscilloscope traces that you label clearly to indicate what they show. Also include the labelled anatomical image you selected in Part 2.
     
  • Post an excerpt (not the whole record) from a direct chart record of the spontaneous activity you recorded. (A chart record where you labeled individual units with numbers would be a good choice.)
     
  • From your computer file of the response to nicotine, create an image of the full experiment to show the overall event, and also use the Zoom window to enlarge several sections to show details. (You can print these as individual items, or you can take screenshots of them to group and label on a single page. Landscape mode will provide the best format for layout.)

Place any remaining data in your group's lab folder (in your drawer).

Clean up

7. Flush the suction electrode with tap water. Turn off power to all equipment, including the DAM-50 preamplifier. Rinse out your dissecting dish, and clean and dry your dissecting tools. Move any stray files from your computer desktop into a folder for today's experiment, and place that folder in the folder for your lab day.