Leech Lab!

During this lab a leech is used to help us better understand the electrical activities of neurons when we used different stimuli and attach them to the skin. Then dyes are injected into the neurons so that we can better see what they look like and how they respond. With a picture of the steps it gives you a better idea of what was being done during the lab.

*Here are the tools that were used in the experiment*

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.

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.

Forceps: Fine forceps for very fine manipulations.

Scissors: Good dissecting angled scissors used here to cut open the body wall.

Pins: Stainless steel dissecting pins for pinning tissue to a dissecting dish or board.

Scalpel: For microsurgery, disposable scalpel blades are better and much more economical than the fixed blade scalpel which needs to be sharpened periodically.

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.

Dissection Microscope: These are binocular microscopes specifically designed for dissection and other micromanipulations. Essentially, it's a high quality high power magnifying glass. You can clearly see individual cells in a leech's nervous system.

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.

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: Medicinal leeches are when they are fully extended, they can reach 15 to 20 cm long. When fully contracted, diameter is roughly 1 ~ 2 cm.

*Definitions of terms*

Action Potential: Also known as nerve impulse or spike. One generally talks about a cell "firing" or "generating" an action potential, or simply "spiking." An action potential is a brief change in membrane potential caused by the rapid opening and closing of transmembrane channels that pass specific ions through. Action potentials travel along axons and transfer information over distance. In this virtual lab, action potentials are generated in many of the cells you can find and appear as an almost vertical line superimposed on a horizontal oscilloscope trace. The total number and the rate of firing of action potentials can encode information, as well as the actual shape of the action potential (some are longer lasting than others, as you can see in the lab).

Amplifier: The amplifier stage is omitted in the virtual lab to simplify the controls, but in reality, between the electrode and the oscilloscope, there will be a special amplifier that costs between $500 to $8,000. This amplifier is designed to specifically deal with an electrode that is very high in electrical resistance, but that's another story.

Anterior: A general anatomical orientation term that means towards the front, which is usually defined as the head of the animal. It can be used as "anterior sucker," meaning the forward sucker, or "The brain is anterior to the gut," meaning the brain is toward the front when compared to the gut.

Opposite to posterior.Axon: A type of a thin tube extending from the cell body of a neurons. Axons are distinguished by their specialization to conduct nerve impulses for long distances, normally in a direction away from the cell body. We say "normally" because it's not always the case. In the touch sensitive cells encountered in this virtual lab, the cell bodies are located in the ganglion, but the action potentials are initiated in the body wall and travel towards the cell body. Also, a neuron can have more than one axon. Again, in our touch sensitive cells, there are axons that disappear from the field of view towards the left and right, in the direction of neighboring ganglia. These axons conduct the touch information to different parts of the nervous system, away from the cell body. Brain: The brain of the leech is a rather simple affair. It is essentially a set of specialized ganglia that have fused to become bigger.

Dendrite: A type of thin tube extending from the cell body of a neuron. Dendrites are specialized to receive information from other neurons, and they do not usually conduct action potentials. In the cells featured in this lab, examples of dendrites can be seen as many finer branches near the center of the ganglion. Dendrites do not transmit information for very long distances, but are instead involved in processing the information they receive from other neurons.

Dorsal: Another anatomical orientation term that means towards the top, e.g. "dorsal fin of sharks." In most animals, the top will be the animal's back. It gets a bit complicated in humans and other bipeds because we move perpendicularly to the axis defined by our backs. Opposite to ventral.

Electrode: Electrodes are what you use here to record the activity of the neurons. The generic term "electrode" is defined as a conductor that is used to establish electrical contact with a nonmetallic substance. So an electrode is what one uses to deliver electric shock to the patient's head in electroshock therapy or to the patient's heart in the emergency room. A glass microelectrode is used in our experiment.

Electrolyte: Ionic solution that conducts electricity. Typically, it's created by dissolving some salt. It is used in the real version of our experiment to fill up the glass micorelectrode. The choice of salt and the techniques developed to fill up the electrode are themselves interesting topics, but a little too esoteric perhaps for this lab.

Electrophysiology: The study of life processes and of the physical and chemical processes involved, particularly the electrical aspects.

Fluorescent Dye: A chemical that emits light when it absorbs electromagnetic radiation from another source. It is often used in neurobiology to view one or a selected number of neurons. The problem is that neurons are usually transparent (unless you are studying the gorgeously colored brain of some sea-slugs). This makes them hard to see. The neurons are also usually packed tightly together, which makes them almost impossible to see individually. Scientists have developed a bag of tricks to overcome this problem, and one of these tricks is to inject (or somehow stain) the neurons with fluorescent dyes. By illuminating the neurons with certain frequencies of light or near-light, the dye "lights up," making the study of their shapes and identities.

Ganglion (pl ganglia): A ganglion is a collection of neuronal cell bodies. In the leech, the central nervous system consists of the brain and 21 segmental ganglia connected by the ventral nerve cord. Each ganglion contains 350 neurons, of which 14 respond to touch and pressure.

Lucifer Yellow: A florescent dye that was introduced in 1978. It is based on the chemical luciferin discovered in 1888 that is present in luminescent organisms such as fireflies. It has proven to be a fiendishly useful product with many applications. In this experiment, it is used in a dissolved form and injected into a neuron, but it has also been used with good effect for immunohistochemistry. Lucifer Yellow fluoresces with bright yellow-green color when stimulated with ultraviolet (wavelength 280 nm) or blue (wavelength 430 nm) light. Because of this, to view the fluorescence, you need an expensive halogen or mercury high-pressure lamp and appropriate filters to block out UV, which can damage your eye.

Microelectrode: An electrode that is very very small. It can be made from a coated tungsten metal or other metal under special fabrication techniques, but in this lab, we use a glass microelectrode. A glass microelectrode is made with a specialized "electrode puller" that heats up the middle of a fine glass tube (usually about 1 mm diameter and about 10 cm in length) and pulls two ends of it apart at high velocity with or without rapid cooling. The result is two electrodes, each with a very fine tip, down to smaller than 0.1 µm depending on the settings on the machine.

Morphology: The form and structure of an organism or any of its parts. Here it is the shape of the neuron. It also refers to the branch of biology that deals in such matters.

Nerve Cord: This is basically a bundle of nerve fibers that connects the ganglia located in each segment to one another and to the brain. In the leech, the nerve cord is located ventrally. It is the leech equivalent of the human spinal cord.

Neuroanatomy: The study of the anatomy of neurons or the nervous system.

Neuron: A nerve cell.

Neurophysiology: A study of the physical and chemical processes of neurons.

Posterior: Opposite to anterior at the rear. Resting Potential: The voltage of a cell at rest.

Segment: The leech is a segmental animal like a caterpillar or an earthworm. Its body is made up of repeating similar units.

Sinus: The circulation system of the leech is a little bizarre. I mean who's ever heard of an animal that sticks its nervous system inside itse blood vessels?? Yet that's exactly how it is. The ventral sinus of the leech is made of a tough fibrous material that appears dark green.

Skin: For convenience, we sometimes use the term "skin" in this lab. But the proper term is "body wall," which is a heavily muscled structure that allows the leech to extend or contract at will.

Ventral: Opposite to dorsal at the bottom.

*Questions from lab*

1. What is the electrode measuring?

Electrodes are what you use here to record the activity of the neurons. The generic term "electrode" is defined as a conductor that is used to establish electrical contact with a nonmetallic substance.

2. Why use leeches in neurophysiology experiments?

Leeches belong to the phylum annelida (worm). The nervous system of the leech consists of the brain, the ventral nerve cord, and ganglia that are located in each segment along the nerve cord. The medicinal leech has 21 segmental ganglia, each containing 175 pairs of neurons. The relatively small number and the large size of the neurons have made leeches favorite subjects of neurobiologists. Most people don’t really care about what happens with a leech and they their neurons are easily accessible.

3. What is the difference between a sensory and a motor neuron.

A sensory neuron by definition is are unipolar nerve cells within the nervous system responsible for converting external stimuli from the organism's environment into internal electrical motor reflex loops and several forms of involuntary behavior, including pain avoidance. In humans, such reflex circuits are commonly located in the spinal cord. Below is a picture of a sensory neuron and its parts. In vertebrates, the term motor neuron classically applies to neurons located in the central nervous system (CNS) that projects their axons outside the CNS and directly or indirectly control muscles. http://wikipedia.org/ Below is a picture of a motor neuron. The website is for both pictures of the neurons.

4. Do you think a leech experiences pain? What is pain?

The leech does not experience pain because it is anestisized. Pain is anything that hurts and no that is not a webster's dictionary definition.

5. What were the two most interesting things about doing this lab?

I did not realize that leeches breathe through there skin and how complex they appear to be inside. I thought it was pretty cool to see the way the different stimulus stimulated the neurons.

6. Anything you found confusing or didn't like about the lab?

No I thought it was pretty self explanatory.

*Procedures*

1. anestisize and desect the leech

2. pin it down in dessection pan and then remove the innards ganglion of the leech

3. cut out ganglion window

4. isolate one ganglion

5. cut the ganglion sinus

6. probe and identify the ganglion sensory cells

7. then pick up each tool and test the cells response

8. then match the cell types!

Above are the different cell types that you can choice from to match up.

This is a picture of the different stimuli that I used and I identified the cell type right.