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Leech Neurophysiology Lab Write Up

January 18, 2012

Conduct this experiment yourself by clicking HERE

 

I. Title: Leech Neurophysiology Lab Write Up

II. Purpose/Objective: Record electrical activities of individual neurons while you deliver mechanical stimulus to the attached skin. Inject flurescent dyes into the neurons to visualize their morphology. Identify the neurons based on the morphology and the response to stimuli, comparing them to previously published results.

III. Hypothesis: If a leech’s skin is stimulated by touch, then if it is touched by a mechanical stimulus the neurons will fire action potentials.

IV. Materials:

Feather: Used to give the leech skin a very gentle touch stimulation. It really doesn’t need to be a feather, it could be q-tips or something. Cost: free.
Probe: A blunt metal rod attached to a wooden handle useful for lifting, pushing, pressing, moving of specimen. Here you use it to lift tissue, and to push the skin as a stimulus. Typical price: $1.00 ~ 10.00
Forceps: Fine forceps for very fine manipulations. The very fine ones are known as Dumont #5 forceps, with tip size of about 0.1 mm X 0.06 mm or smaller. Typical price: $15.00 ~ 45.00
Scissors: Good dissecting angled scissors used here to cut open the body wall. Teaching scissors are cheaper, but some ultra-fine dissecting scissors could cost upward of $400, and you better not drop that, because once you drop it, chances are, it’s ruined. Typical price: $15.00 ~ 60.00
Pins: Stainless steel dissecting pins for pinning tissue to a dissecting dish or board. You can drop these and not worry about it. $1.00
Scalpel: For microsurgery, disposable scalpel blades are better and much more economical than the fixed blade scalpel which needs to be sharpened periodically. Blade: $0.50 Handle: $10.00 Used here to cut all kinds of things.
Dissection Tray: A tray half-filled with hard wax so that you can stick pins into it to stabilize specimen for dissection.
Leech Tank: Leeches are kept in pond-water (you can actually buy an instant pond-water mix to add to tap water.) If kept in a refrigerator, they can stay happy in it for weeks at a time without feeding.
20% Ethanol: Used to anesthetize the leech. Besides being more humane, it has the added benefit that it stops them from moving, making it easier to pin down the leech.
Leech Tongs: These are basically gross anatomy forceps with blunt tips so that you will not harm the leech as you pick it up. Maybe about $ 10.00
Dissection Microscope: These are binocular microscopes specifically designed for dissection and other micromanipulations. Essentially, it’s a high quality high power magnifying glass. The price varies on quality and if you’ve looked through binoculars of different quality, you can appreciate what a difference good optic makes. On a good one, you can clearly see individual cells in a leech’s nervous system. Cost about $1,000.00 ~ $7,000.00
Micromanipulator: A device used to position items with sub-micrometer precision in three dimensions. Here we mount our electrode on it to guide it accurately to a neuron. For work on a leech, a mechanical manipulator would suffice which is about $700.00. More accurate hydraulic or electronic ones may cost up to $10,000.00
Oscilloscope: Basically a sophisticated voltmeter. What you see on the screen is a real time display of voltage (vertical) plotted against time (horizontal). Useful because voltmeters can’t track rapidly changing voltages, and even if they could, you couldn’t read anything. Cost $2,000.00 and up.
Leech: Medicinal leeches are about $15.00 each. When fully extended, they can reach 15 to 20 cm long. When fully contracted, diameter is roughly 1 ~ 2 cm.

V. Procedure:

  1. Pick up the leech with the tongs and anethetize the leech in the 20% ethanol solution.
  2. Next pin the leech dorsal side up through the anterior and posterior suckers onto the dissection tray, stretching the leech in the process.
  3. Using the scissors, make a cut along the mid-line of the dorsal side of the leech. Be careful not to damage any deep structures. Using the forceps spread the leech apart and pin back the leech.
  4. With the probe remove all internal structures to expose the ventrally located nerve cord. The leech’s nervous system is encased within the ventral sinus, which is a dark green color.
  5. Using the dissecting microscope notice that there are many swellings up and down the sinus. These swellings contain segmental ganglia. Using and the scalpel cut a window in the body wall underneath the ganglion, making sure not to damage the nerve cord or any other nerves in the process. Once the window is cut remove the body wall with the forceps.
  6. Isolate the section with the window by making two parallel cuts across the animal. With forceps, flip the piece of skin over and pin the skin down.
  7. Next, using the dissecting microscope cut the sinus with a scalpel and using forceps, tease apart the sinus to expose the ganglion. Individual cells should now be able to be viewed under the microscope.
  8. Use the micromanipulator tip from the micromanipulator to probe a cell. Once you find a cell use each tool (feather, probe, and the forceps) to test the cell response. Also make sure to dye each cell using the dye injection and click the UV switch to view the dyed cell. Then use the atlas to compare your data to identify which cell you have located.
  9. Continue step 8 until you have identified all five cells.

VI. Data:

  • The data  collected from using the micromanipulator should look exactly the same as your atlas if you find all five cells. 

VII. Conclusion: 

After conducting this experiment my hypothesis did prove to be correct. When you touched a neuron with a mechanical stimuli such as the feather, probe, or forceps they did in fact fire action potentials.

From this experiment I learned that  there are different types of cells that react differently to each stimulus. For example, if you look at the data you can see that a feather causes for no change in an N, P, or X cell, but when it is used on a T or R cell there is activity. I also learned that some cells such as the R cell are firing action potentials without any stimulus and others such as the X cell very rarely send action potentials.

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