So in Science(or maybe only in science with Mr. Ludwig) you don't always do conduct a lab in person. To learn about Neurophysiology our class did an online virtual lab that involved leeches.(I'm actually kind of glad it was an online lab!)
Objective:
In this lab we wanted to record electrical activities of individual neurons while delivering mechanical stimulus to the attached skin and inject fluorescent dyes into the neurons to visualize their morphology. We also wanted to identify the neurons based on the morphology and the response to stimuli, comparing them to previously published results.
(Some quick definitions to help you understand this experiment!
-Neurophysiology: A study of physical and chemical processes of neurons
-Neuron: A nerve cell
-Morphology: The form and structure of any organism or any of it's parts. ~In this experiment it is the neuron.
Materials:-Feather: This is used to give the leech skin a very gentle touch stimulation
-Probe: This is a blunt metal rod attached to a wooden handle and in this experiment ou use it to lift tissue, and to push the skin as a stimulus.
-Forceps: These are fine forceps for very fine manipulations.(also known as Dumont #5)
-Scissors: These need to be good dissecting angled scissors. For this experiment they cut open the body wall.
-Pins: These are stainless steel dissecting pins for pinning tissue to a dissection dish.
-Scalpel: For this experiment you need disposable scalpel blades because they are better for microsurgery.
-Dissection Tray: This is a tray that is half-filled with hard wax so that you can stick pins into it to secure the leech for dissection.
-Leech Tank: For this experiment it is best to keep leeches in pond water.
-20% Ethanol: This is used to anesthetize the leech. (Time for the first FUN FACT of the semester!: 20% ethanol is humane and stops the leech from moving, which makes it easier to pin it down.
-Leech Tongs: These are basically gross anatomy forceps and are used to pick up the leech.
-Dissection Microscope: This is a special microscope designed for dissections. It is basically a high quality high power magnifying glass.
-Micromanipulator: This is a device that is used to position items with sub- micrometer precision in three dimensions. In this experiment we mount our electrode on it to guide it accurately to a neuron.
-Oscilloscope: This is basically a sophisticated voltmeter. [On the screen you see a real time display of voltage(vertical) plotted against time(horizontal)]
-Leech: The thing we are dissecting!
Procedure:
1. First we caught the leech and anesthetized it in 20% ethanol solution.
2. We pinned the leech to the dissection dorsal(the top) side up. We stretched the animal out in the process.
3. Using the scissors we cut the skin along the mid-line on the dorsal side. We were very careful not to damage the deep structures. Then we used the forceps to carefully peel apart the skin and pin down each side of skin. (FUN FACT: DOing this exposes the innards of the leech, including the digestive, excretory, and reproductive organs!)
4. Next we carefully removed the gut and the other internal structures so we could see the ventral nerve cord.
5. We moved the leech under the Dissection microscope so we could look at the ganglia of the nervous system. (a collection of neuronal cell bodies. Using the scalpel we cut a window along the sinus(circulation system of a leech) under a ganglion. We made sure not to do any damage to the nerves.
6. Using the scalpel we cut a section of ganglion out of the leech making two parallel cuts that were perpendicular to the anterior-posterior axis. Then using forceps we flipped the piece of skin so that the outer skin was facing up and pinned the skin down.
7. Under the dissection microscope we cut the sinus with an ultra fine scalpel and using fine forceps we carefully peeled apart the sinus to expose the ganglion. We could then see individual cells under the microscope.
8. We moved the skin under the Micromanipulator to find the cells. We used the electrode to gain control of it and moved the electrode any place over the ganglion to stimulate penetrating the cell. (this is actually much harder in real life) We watched the oscilloscope so we could see when we had found a cell. The oscilloscope turned the signals from the cell into a sound wave display. Then we used a feather, probe, and forceps to see if the cell would respond to see any stimulus. When the cell showed a spike it responded in firing an action potential. Then we compared our data to the data in the atlas. Then we dyed the cells with the fluorescent dye to see where the different cells were located.
9. Next we used the UV Switch to see the dye. This let us see the structure of the cells.
10. At the end we used the electrophysiological and neuronanatomical data to identify each cell. After we identified one cell we went back and identified the other 4 cells using the same process.
Analyzing Data:
All together I identified 5 cells. I could tell what was each cell by how it responded to the different stimulus.
The atlas above is what I used to compare my results to. In this experiment I learned how the different cell types react to the different stimulus. For example cell type N only reacted to the forceps while cell type P only reacted to the probe. Some cells, like cell type R, fire action potentials without any stimulus affecting them and some cells, like cell type X, don't react to any stimulus at all. Cell type T responded differently to each of the different stimulus.
Conclusion:
This experiment taught me a lot about the body. There are so many different types of cells in one small ganglion. (a collection of neuronal cell bodies). All the cells are different. They each respond differently to the different stimuli. This virtual lab helped me understand the different types of cells and how they are different.
If you would like to try this online virtual lab follow this link! (I suggest it!)
http://www.hhmi.org/biointeractive/vlabs/neurophysiology/index2startlab.html
