During this lab we dissected a leach to test and observe the nervous system. The equipment for this lab were: Feather,Probe,Forceps,Scissors,Pins,Scalpel,Dissection Tray,Leech Tank,20% Ethanol Leech Tongs, Dissection Microscope,Micromanipulator,Oscilloscope, and finally of course the leech.
Step 1
Catch and anesthetize the leech in 20% ethanol solution. Ethanol is not an anesthetic for vertebrate animals, but can be an effective anesthesia for small creatures that breathe through the skin like the leech. Like in many things, too high a concentration will be harmful or fatal.
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
Using scissors, make a cut in the skin along the mid-line on the dorsal surface, taking care not to damage deep structures.
Using forceps, carefully tease apart the skin along the cut and 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 exposes the innards of the leech, including the digestive, excretory and reproductive organs. You cannot see the nervous system yet, because they are located ventrally.
Step 4
Carefully remove the gut and other internal structures to expose the ventrally located nerve cord. The nervous system of the leech is encased within the ventral sinus, which is dark green in 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 nerves in the process.
Step 6
Isolate a section of the animal by making 2 parallel cuts across the animal (perpendicular to the anterior-posterior axis), but sufficently separated so that the strip you remove contains at least one ganglion.
Then, with forceps, flip the piece of skin over so that the outer skin is now face up. Pin the skin down. If you don't know why you are doing this, go read the Why are we doing this? of Step 5 and come back.
Step 7
Cut the sinus with an ultra fine scalpel and using fine forceps, carefully tease apart the sinus to expose the ganglion. Individual cells can now be viewed under the microscope.
In reality, you would only use the scalpel here only if you are extremely good at microdissection. It's very difficult to cut just the sinus without accidentally damaging the ganglion underneath, but hey, we are all perfect in cyberland. Normally, this is done with a pair of very fine forceps.
Now you've come to the crux of the matter. All the preparation so far has been to make this step possible. You might want to review Nervous System background or Electrical Equipment background at this point.
Click on the electrode to gain control of it. Move the electrode to somewhere over the ganglion then click on the mouse button. This simulates the process of penetrating the cell, which is much more demanding in reality (see "What it's like in reality." for details). Keep your eyes glued to the oscilloscope display while you are doing this. If you find a cell, the display will change. If you see no change, then you have not found a cell. Keep moving your electrode around and clicking until you find a cell. The sound you hear is the oscilloscope display you are seeing fed into an audio amplifier. It provides an audio feedback to what you see on the screen.
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 response. The cell may fire action potentials or spikes. The response characteristics will be used when you are comparing your data with published data compiled in the 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 the electrode that resulted in the ejection of Lucifer Yellow from the tip of the electrode into the intracellular space. Lucifer Yellow will passively spread throughout the cell after a while. Now you can turn on ultraviolet (UV) light by pushing "UV Switch.". Lucifer Yellow fluoresces bright yellow-green under UV and you will be able to visualize the cell in question, including its axon, dendrites, cell body and so on.
Step 10
You now have electrophysiological data and neuroanatomical data from your experiment. Try to identify the cell based on published data (Atlas) There are many cells in different locations of this ganglion. Repeat the whole procedure for as many cells as you would like.
I showed the steps above just to show the exact steps I went through to complete the lab and see how the nervous system properly works. In this lab after taking all of the insides of the leech we found the central nervous system of the leech. Then we cut parts of the nervous system into pieces and tested them to see what kind of cells they are. At the end of the lab when you dye the cells and probe them to see what kind of cell in the nervous system you are testing. Certain cells did not stimulate when poked by the feather while others one were very stimulated by it. This Lab showed me how the different cells come together to form the nervous system. Also after doing this lab I got to see a new type of technology with the Oscilloscope. I have never even heard of the Oscilloscope but after using this lab I got to see what it can do. After taking the nervous system apart piece by piece I was able to see how amazing and mind blowing the nervous system is when it is all put together and functioning properly. Even though this lab was rather simple I believe I learned a lot about the nervous system.
