Leech Neurophysiology Lab

Neurophysiology Lab


Purpose: To observe electrical activity in a Leech neuron.



Hypothesis: My hypothesis is that the electrical activity in a leech neuron is going to be really high, just because of what they are capable of doing.

Materials: The materials include: Feather, probe, forceps, scissors, pins, scalpel, dissection tray, leech tank, 20% Ethanol, leech tongs, dissection microscope, micromanipulator, oscilloscope, and a leech.


Procedure:
              Step 1) Catch and anesthetize the leech in 20% ethanol solution.

              Step 2) Pin the animal dorsal side up through the anterior and posterior suckers onto a dissection tray, stretching the animal in the process.

              Step 3)Then get the scissors, make a cut in the skin along the mid-line of the dorsal surface, but not to deep so won't damage anything in the inside of the leech. Secondly, you get the forceps and carefully tease apart the skin along the cut, then pin down the left and right halves of the skin to each side, so that the leech is pinned open with the inside of the skin facing up. This will then uncover the innards of the leech, including the digestive, excretory and reproductive organs. You will not yet see the nervous system.

              Step 4) Carefully remove the gut and other internal structures to see the ventrally located nerve cord. The nervous system of the leech is encased within the ventral sinus, which is the dark green color.

             Step 5) Notice that there are many swellings up and down the sinus. These contain the segmental ganglia of the nervous system. To make one of them accessible, first we cut a window in the body wall underneath a ganglion, taking care not to damage the nerve cord or any attached nerve in the process.

             Step 6) Isolate a section of the animal by making two parallel cuts across the animal, but sufficiently separated  so that the strip you remove contains at least one ganglion.
Then with the forceps flip the piece of the skin over so that the outer skin is now face up. Pin the skin down.
             Step 7) Cut the sinus with an ultra fine scapel and using fine forceps, carefully tease apart the sinus to expose the ganglion. Individual cells can now be viewed under the microscope.
           
            Step 8) Now using a feather, probe, or forceps, push around the skin of the animal. Observe if the cell you have penetrated responds to weak (feather), medium (probe), strong (forceps), or any stimulus. Note the pattern of the response. The cell may fire action potentials or spikes. The response characteristics will be used when you are comparing your data with published data complied int he atlas. When you are satisfied with the electrophysiology, you can start the anatomical investigation by injecting the cell with a fluorescent dye. Push the button labeled "Dye Injection."
          
          Step 9) Next, we will visualize the morphology of the neuron from which you have just recorded using a fluorescent dye. having pushed the button labeled "dye injection" the amplifier system has passed an electric current from electrode that resulted in the ejection of the lucifer yellow from he tip of the electrode into the intracellular space. Lucifer yellow will passively spread throughout the cell after a while. Now you can turn on UV light by pushing "UV switch". Lucifer yellow fluouoresces bright yellow-green under UV and you will be able to visualize the cell.     



Results: The results that I have gotten from this lab is that in order to find out what exactly is going on with the leech and how their whole neuron system, you have to go in and take everything out and like actually test out their sinus and ganglion, so on and so forth. So it was really neat how you would have to do that!
Conclusions: Only some cells were sensory to some items and not all. They  can only effect some part of the leech's body in order to effect it!