Alzheimers

As a group, we recently created and presented a Prezi for the class.  The base of our project surrounded neurons and the brain and how that is related to alzheimers.  Our work can be seen here.  

http://prezi.com/ehnj2if1x4ud/alzhimers-cant-spell/

Neurophysiology Lab

Objective Record electrical activities of individual neurons while you deliver mechanical stimulus to the attached skin. Inject flurescent dyes into the neurons to visualize their morphology. Identify the neurons based on the morphology and the response to stimuli, comparing them to previously published results.

Equipment List
	Feather: Used to give the leech skin a very gentle touch stimulation. It really doesn't need to be a feather, it could be q-tips or something. Cost: free.
	Probe: A blunt metal rod attached to a wooden handle useful for lifting, pushing, pressing, moving of specimen. Here you use it to lift tissue, and to push the skin as a stimulus. Typical price: $1.00 ~ 10.00
	Forceps: Fine forceps for very fine manipulations. The very fine ones are known as Dumont #5 forceps, with tip size of about 0.1 mm X 0.06 mm or smaller. Typical price: $15.00 ~ 45.00
	Scissors: Good dissecting angled scissors used here to cut open the body wall. Teaching scissors are cheaper, but some ultra-fine dissecting scissors could cost upward of $400, and you better not drop that, because once you drop it, chances are, it's ruined. Typical price: $15.00 ~ 60.00
	Pins: Stainless steel dissecting pins for pinning tissue to a dissecting dish or board. You can drop these and not worry about it. $1.00
	Scalpel: For microsurgery, disposable scalpel blades are better and much more economical than the fixed blade scalpel which needs to be sharpened periodically. Blade: $0.50 Handle: $10.00 Used here to cut all kinds of things.
	Dissection Tray: A tray half-filled with hard wax so that you can stick pins into it to stabilize specimen for dissection.
	Leech Tank: Leeches are kept in pond-water (you can actually buy an instant pond-water mix to add to tap water.) If kept in a refrigerator, they can stay happy in it for weeks at a time without feeding.
	20% Ethanol: Used to anesthetize the leech. Besides being more humane, it has the added benefit that it stops them from moving, making it easier to pin down the leech.
	Leech Tongs: These are basically gross anatomy forceps with blunt tips so that you will not harm the leech as you pick it up. Maybe about $ 10.00
	Dissection Microscope: These are binocular microscopes specifically designed for dissection and other micromanipulations. Essentially, it's a high quality high power magnifying glass. The price varies on quality and if you've looked through binoculars of different quality, you can appreciate what a difference good optic makes. On a good one, you can clearly see individual cells in a leech's nervous system. Cost about $1,000.00 ~ $7,000.00
	Micromanipulator: 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. For work on a leech, a mechanical manipulator would suffice which is about $700.00. More accurate hydraulic or electronic ones may cost up to $10,000.00
	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. Cost $2,000.00 and up.
	Leech: Medicinal leeches are about $15.00 each. When fully extended, they can reach 15 to 20 cm long. When fully contracted, diameter is roughly 1 ~ 2 cm.

Procedure

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 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 (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. Go to the Atlas page to identify the cell.  Results Review Really, I thought this was a nice way to perform a dissection based lab.  I don't have serious issues with dissections but it's always been rather unsettling, cutting up and poking around in dead things.  It was nice going step by step with the computer so as not to forget anything,  which we seem to do rather often in real labs.  It was also nice to have explanations as to what exactly each step meant and what we were supposed to be getting out of it.  I didn't do extremely well at the quiz section at the end mainly because my computer refused to give me proper options....still unsure as to why this happened.