Tuesday, January 24, 2012

Leech Neurophysiology Lab Write Up

Introduction to the Neurophysiology Leech Lab  
Our class followed an online virtual direction of a leech (we did this virtual lab, rather than in the classroom, because all of the material is too expensive and the precise procedures on such a small area would be hard without training and experience). This was to demonstrate and educate us on neurophysiology. Our objective was to record electrical activities of individual neurons while we delivered mechanical stimulus to the attached skin, inject fluorescent dyes into the neurons to visualize their morphology, and identify the neurons based on the morphology and the response to stimuli, comparing them to previously published results.
     
There were many materials used in this lab:
  • pins
  • scalpel
  • dissection tray
  • probe
  • forceps
  • feather
  • scissors
  • leech tank
  • 20% Ethanol
  • leech tongs
  • dissection microscope
  • micro-manipulator- 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.
  • 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. 
  • leech
Procedures:


Step 1

We caught and anesthetized the leech in a 20% ethanol solution.

Step 2 

We pinned the leech's dorsal side up through the suckers onto a dissection tray and stretched the animal open.


Step 3

We used scissors to make a cut in the skin along the mid-line on the dorsal surface. Then we used the forceps to pull the skin back from the incision and pinned that skin down so that we could see the structures on the inside of the leech.

 

Step 4

We carefully removed the gut and other internal structures to expose the nerve cord. 




 

Step 5

We then noticed the swellings on the sinus which contained the segmental ganglia of the nervous system and we cut a small piece in the body wall under the ganglion.

 

Step 6

We then made 2 parallel cuts across the leech and turned it skin side up and pinned it down.

 

Step 7

We cut the sinus with a scalpel and used the forceps to expose the ganglion and then we looked at it under a microscope (Clearly we couldn't dissect something this small so the cyberlab is great for steps such as these).

 

Step 8

We used the electrode over the ganglion to simulate the process of penetrating the cell. We then looked at the oscilloscope to tell us if we found a cell. The oscilloscope turned the signals from the cell into an sound wave display. We used the feather, probe, and forceps and watched how the cell responded. If the cell shows a spike then the cell responded in firing an action potential. We then compared our data to that in the atlas. We then dyed the cell with fluorescent dye to see where the certain cells were located.
For background info on this step you can check out these links!
Nervous System background
Electrical Equipment background

 

Step 9

Then we used the UV Switch to see the dye which allows us to see the cells structure.

 

Step 10

We then used the electrophysiological and neuroanatomical data to identify. After we finished this step for one cell we went back and repeated the procedure for all 5 different cells.

Data and Analysis:

This is what I identified as a type x-cell. I identified it as this type because of its position, structure, and its stimuli. The type x-cell recorded no stimuli when touched or poked with the stimulus tools.



This is what I identified as a type p-cell. I identified it as this type because of its position, structure, and its stimuli. The type p-cell recorded stimuli when poked by the probe. 



This is what I identified as a type n-cell. I identified it as this type because of its position, structure, and its stimuli. The type n-cell recorded stimuli when poked by the forceps.



This is what I identified as a type r-cell. I identified it as this type because of its position, structure, and its stimuli. The type r-cell had a constant record of stimuli even when it was not touched by any of the tools.



                                                                                                                  
This is what I identified as a type t-cell. I identified it as this type because of its position, structure, and its stimuli. The type t-cell recorded multiple types of stimuli. When touched by the feather the cell reacted a lot, the probe a little less, the forceps a little less, and finally, when nothing is touching it, no stimuli.



This is the atlas that I referred to help me determine the cell type of my data.


Conclusion:

After I identified each of the cell types I got a Congratulations note and this picture showed me all the placements where the cells could be found. This picture wrapped up all of the types of cells and helped me to know where to place them in the ganglion. It was interesting to learn that each of these types responded to different stimuli and that they each had thier own certain region. In the end, it all made a lot of sense as to why our bodies work the way they work. It is amazing how small of detail our bodies, along with others such as leech's, come down to. This lab really helped me to better understand the cell types and neurophysiology plays a huge role in our bodies.