Hearing Project*

leech lab*/ Neurophysiology

Objective:  
This lab was to identify,analyze a neuron. Also 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. This was also to help us to learn neurophysiology- study of physical and chemical processes of neurons.


Materials:

leech

scalpel

pins

dissection tray

probe

forceps

feather

scissors

leech tank

20% Ethanol

tongs

dissection microscope

micro-manipulator

Oscilloscope

Steps:

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

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.

Step 8

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. 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.


This lab taught me how to identify the different cell types and how the cells  countered to different stimuli.

http://www.hhmi.org/biointeractive/vlabs/neurophysiology/index2startlab.html