Heart Breaker

We dissected a heart this past valentines day, a bit ironic i know. In this lab, we had to measure some of the more...important parts of the heart. We measured the diameter of the Aorta, Pulmonary Trunk, right and left atrium, right and left ventricles, and the outer walls. We measured all of those things on 3 different species' hearts. There was the Sheep heart, Pig heart, and the Cow heart. Though the basic structure of these hearts were pretty much the same, the major difference in them came in the overall size of the parts we had to measure. For instance, the Sheep heart was no bigger than ones hand, where as the cow heart was massive! So you could conclude that it was easier to find things like the Aorta on the cow heart than the sheep heart. The reason for this extreme size difference in the hearts, could be contributed to the amount of blood that needs to be pumped into the body, so a cow needs to have more blood going through their body than a sheep needs to go through theirs. An interesting fact about the pig heart, is that it is about the same size as a human heart. Therefore the human and the pig need about the same amount of blood pushed throughout their bodies.

One of the questions that we were asked to find out by using the microscope and analyzing parts of the veins outer wall, and an arteries outer wall, was what is thicker, the wall of the veins or the walls of the arteries. The answer is the walls of the arteries, because they have more blood flowing through them at a faster rate than veins do. As for the Cardiac muscle (around the heart), it is a striated muscle, where as most other muscles are silky smooth. After viewing that, we went ahead and looked at a coronary artery with atherosclerosis, which is a kind of plaque that builds up in some arteries, which can then restrict the blood flow, and possibly cause a blood clot.

The sheep heart had the largest right side outer wall at 5 cm, and the pig heart has the largest left side outer wall coming in at 4 cm. In terms of overall size, the cow heart is the largest, yet in terms of the part, the cow heart is only the biggest in the left ventricle. I find that to be somewhat...odd.

brain dissection

We did a lab where we got to cut apart a brain and identify the parts that help to give the brain is funky looking form. For safety reasons we had to use rubber gloves, and an optional goggle accessory. We started off the lab by observing the Dura mater, or outer meanings. though not all the brains had them, the one that we had did. se we wound up cutting of this weird looking white layer from the outside of the brain. After that, we had to identify the cerebrum, where we saw the groves that are known as sulci. you should also see the ridges called gyri, which are pretty close to the medial longitudinal fissure, which separates the left and right hemispheres of the cerebral cortex. We then had a little bit to go and identify nearly every part of the brain. from the thalamus to the central canal, and from the spinal cord to the olfactory bulb.

We wound up getting to cut the brain horizontally. Which is the horizontal way to cut the brain, you just put it on its side, and cut through it. It leaves you with some pepperoni looking slices in your dissection tray. The odd clear stuff on the far left side of the picture is the Dura matter, which is a white "casing" that the brain happens to be in...and its pretty grodi.

Virtual Leech Dissection

http://www.hhmi.org/biointeractive/vlabs/neurophysiology/notebook_nav/noteframe.html

Introduction

Selecting the Notebook tab displays the laboratory protocol in this window. Also, when the main lab window loads another portion of the exercise, appropriate section of the protocol is automatically displayed. Some words show up as hyperlinks like this. That means that more information is available as either a glossary entry or background. Also, each piece of equipment is described in the equipment section. 

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 theanterior-posterior axis), but sufficently separated so that the strip you remove contains at least oneganglion.

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.

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 afluorescent 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 theelectrode 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.

In this lab, you get to dissect a virtual leech which between me and you, it definitely interesting. It is a very interesting thing, and you learn a lot. Especially considering that you get to basically, electrocute the leech. Though it is a little bit difficult to navigate around their website, it is still a good learning experience.