PNB 2264 Lab 8: Earthworm Action Potentials

Intracellular recording

Involves measuring voltage and/or current across the membrane of a cell, fine sharp glass mini electrode inserted into the cell

Extracellular recording

Detects only small potential differences that arise from action current flowing in the extracellular medium around the nerve fiber, changes at membrane surface as opposed to across membrane

What is the relationship between stimulus strength and response amplitude in a single axon?

As long as the stimulus is strong enough to stimulate an action potential, the response amplitude will reflect an action potential propagation, which reflects the "all-or-none" principle of action potentials. It does not matter how weak or strong the stimulus is as long as it brings the axon to threshold to fire that action potential

The typical shape of the action potential recorded in this lab is biphasic with two peaks of opposite sign. Can you explain why this is? (hint: this question is asking in the context of extracellular recording.)

The experiment was set up in a way where we inserted two electrodes into the worm, one negative and one positive, and took measurements from these two points. When the action potential first reached the negative electrode, the graph dipped downwards because of the opposing like charges. When the action potential then reaches the positive electrodes, the graph shoots back up because of the opposite charges attracting each other

Describe in detail the cellular events responsible for the absolute refractory period AND the relative refractory period

In absolute refractory period, the Na+ channels are inactivated COMPLETELY, so sodium cannot enter the cell and depolarize it to bring it back to baseline (around -70 mV). In relative refractory period, some of those Na+ channels are open and activated, and if enough positive charge can get back into the cell, another action potential can be fired, hence the name "relative."

Biphasic recording

Both positive and negative deflection on the graph

- Intracellular recordings
Positive deflection (depolarization) and negative deflection (hyperpolarization)

- Extracellular recordings
Positive deflection (when reaching negative electrode) and negative deflection (when reaching positive electrode)

Absolute refractory period

Majority of sodium channels remain in an inactive state, no stimulus can trigger another action potential

Relative refractory period

Mixed population of inactive and closed sodium channels, action potential can still be triggered

Location of electrodes

Stimulus at beginning, followed by negative electrode, and then positive electrode

Difference equation (solving separately for each axon, two trials of the same axon)

V= (D2-D1)/(LP2-LP1)

Stimulus artifact

Has a negative and positive inflection, NOT the action potential

First response after stimulus artifact

Medial axon, has larger diameter, faster velocity, threshold of voltage needed for response is lower, latency period is shorter

Second response after stimulus artifact

Lateral axon, smaller diameter, slower velocity, threshold of voltage needed for response is higher, latency period is longer

What has occurred if you only see one response after the stimulus artifact?

The voltage given was only strong enough to produce a response from the lower-threshold medial axon and not the higher-threshold lateral axon

Where do you put the marker and point range when looking for latency?

Marker before the first dip in the stimulus artifact, point range ending at beginning of axon response

Absolute method

V = D/LP

Calculating distances

From stimulus to given electrode

Worm lab anesthesia step one

1. Place worm in dish DORSAL SIDE UP. Fill the dish with sufficient ethanol/earthworm Ringer's solution to cover the worm, leave in for approximately five minutes

Worm lab anesthesia step two

2. Test if worm is sufficiently anthesthetized by prodding it gently with blunt instrument

Worm lab anesthesia step three

If worm doesn't react to touch, it is ready to use. If still reacts, leave in solution for 3-5 more minutes

Worm lab placement step one

Anesthetized worm should be laid flat out and straight on dissecting tray, about 50 mm from one edge

Worm lab placement step two

To keep worm anesthetized, apply a few drops of ethanol/Ringer's solution along its length, soak up excess with tissue, too much alcohol will interfere with recording

Worm lab electrode setup step one

Lay the head end on the stimulating electrodes as shown, make sure two wires of the stimulating electrodes touch the worm

Worm lab electrode setup step two

Place three needle electrodes into the body of the worm, just off center line to avoid damage to nerve cord

Worm lab electrode setup step three

Pin attached to green wire is ground electrode, the pin attached to the black wire is the positive electrode, pin attached to white wire is negative electrode

Worm lab electrode setup step four

Ground wire should be closest to the stimulus electrode. Position negative recording electrode 2-3 cm away from ground electrode, put positive/negative electrode 5-10 mm apart, NO WIRE touching another wire

Potential difference equation

Charge @ positive electrode - charge @ negative electrode

Stimulating electrode should be placed at...

Darker area of the worm on the dorsal side, anterior

Where should needle electrodes be placed?

Off-center as to not hit any nerves in the center of the earthworm, the ground wire should be closest to the stimulus electrode

Which fiber has the higher conduction velocity, median giant or lateral giant?

Median giant fiber

What can contribute to differences in response and conduction velocity?

Temperature, myelination, axon diameter, ion concentrations, ion channel density