THE MUSCLE LAB

Goals
(1) You should become versatile with the use of the electrophysiological computer accessories
(2) You will achieve the above in the context of learning muscle physiology.

Review

[It is presumed that you have this background from Introductory and Cell biology, but, if not, here is my muscle lecture outline from the last time I taught BL A-106 (Principles of Biology II)]

Molecular biology of muscle contraction:
(1) Calcium from sarcoplasmic reticulum binds troponin and causes tropomyosin to come off actin's myosin binding sites.
(2) ATP breaks the actin-myosin bond, myosin phosphorylation as ATP->ADP rachets the myosin head, myosin rebinds actin, and finally release of inorganic phosphate by myosin causes the myosin head to bend and hence the sliding of myosin relative to actin (ATP breaks the bond again).

Excitation-contraction coupling:
(1) An action potential in the spinal motor neuron goes to the neuromuscular junctions of all the muscle fibers (cells) in the motor unit (all the striated muscle cells innervated by one spinal motor neuron.
(2) Vesicles of acetylcholine are released (this from my neuro course motor outline) and bind to nicotinic acetylcholine receptors (channels) on the end plate.
(3) Depolarization at the end plate leads to a spike on the muscle fiber's (cell's) membrane.
(4) T-tubules (transverse tubules) bring the action potential near each sarcomere in each muofibril.
(5) Dyhydropyridine (blocking drug) receptors in t-tubule couple to...
(6) ...ryanodine receptors (calcium channels) in sarcoplasmic reticulum.

First little exercize
I found some of those little rubber hammers. So let's all try the knee-jerk (patellar) reflex. Find the monosynaptic reflex on p. 430 (Fig. 13-1, the top part). Tap GENTLY on the tendon.

Today's lab:
(1) This will be our first use of the PowerLab for recording (except for one exercize in Electrophysiology)
(2) We will stimulate the muscles (and hopefully the nerve) using the stimulator of the PowerLab.
(3) We will measure strong responses with the hand dynamometer feeding into the PowerLab.
(4) We will measure finger twitches with the force transducer feeding into the amplifier which, in turn, feeds into the PowerLab.
(5) You should become versatile with the use of the electrophysiological computer accessories.

Today's lab uses human subjects but is otherwise much like the classic "frog nerve-muscle" preparation, one of the labs at University of Missouri - Columbia. Electrodes in the gastrocnemius muscle (right) stimulate the muscle directly while a cuff on the sciatic nerve (left) stimulate the muscle via the nerve. One tendon is fixed down while the other is tethered to the force transducer.

Directions:

[Since this was done in the computer lab Fall, 2004, this is just included for review.]

Preliminaries:

(1) The amplifiers have no on-off switch, so we handle that by plugging them in.
(2) These PowerLabs were purchased when input of such an interface into the computer was via SCSI, and the general rule is that the SCSI device should be on before the computer (not hot-swapable like USB).
(3) After the PowerLab is on, turn on the computer with the tower or the keboard button.

Specifics:

Recording with the hand dynanometer:

(1) Pull down under the Apple (upper left) to !Chart (where we put the alias for Chart v4.0.1 for PowerLab [AD Insruments]).
(2) If a blank record appears, close it with a click to the box in the upper left (or Close under File)
(3) Under file, pull down Open, and, if you're lucky, it'll default to a folder with the PowerLab experiments.
(4) Choose Muscle #1
(5) With only the Dynanometer feeding into Channel 1 on the PowerLab...
(6) ...start the recording and squeeze the ball and stop the recording to note that the record is in range.
(7) Note that the sensitivity defaults to 200 (100 is too high and goes off scale when squeezed, and 500 is off scale)
(8) Have fun testing the grip of the lab partners.

Stimulation:

Caution:
The volunteer will get mild electric stimulation on one arm. (1) Nobody with heart or nerve problems should volunnteer. (2) Discontinue if it is bothering you. (3) Don't apply the stimulation across the body.

(1) Under Set-Up, pull down isolated stimulator panel
(2) The stimulator switch on the PowerLab must be on
(3) Put an electrode on the back of the wrist and attach the red snap lead
(4) Stimuli will only output when you start the record; note that a light on the PowerLab, as well as an upward deflection on the bottom trace, marks the stimuli.
(5) Be prepared to change the mA and the frequency of stimulation.
(6) Touch various places on the front of the forearm gently with the black probe and note that specific muscles can be made to twitch.
(7) Pressing harder, see if you can find a place where a forceful dynanometer recording is registered. [This would indicate a stimulation of the nerve.]

Alternate - recording with the force transducer

[Since this was done in the computer lab Fall, 2004, some of this this is just included for review.]

(This can be done for small finger twitches, if you could not achieve a forceful squeeze [#7 above], based on stimulation of one of the muscles)
(1) Uuit Chart then launch it again. Note there are 8 channels, the bottom 4 of which are bogus since there are only 4 inputs, and the bottom 4 are turned off.
(2) Turn off 2, 3, and 4 and pull down the bottom lines to the bottom to fill the screen with channel 1.
(3) Feed the force transducer into the bridge amplifier (x1 gain will work), and feed that into the PowerLab (gain of 2 will be sensitive).
(4) Note that the trace is likely off scale - and here the position on the bridge helps a lot. It's a 10 turn pot (potentiometer = variable resistor).

We can calibrate the force transducer

[Since this was done in the computer lab Fall, 2004, this is just included for review.]

(1) Note that the ordinate is in mV
(2) Change the sensitivity to 5 mV
(3) Start the record (on screen), put on a 1 g weight, note the deflection, stop the record.
(4) Set the cursor on the low and high and note that you can see the reading
(4) Pull down under units of conversion, on two point conversion, set your low and high readings on the left and 0 and 1 on the right [then select g].
(5) Click OK and note that the ordinate is now in g

Procedures
(1) After getting a reproducible reading with one or both of the above,...
(2) Change the amplitude (mA) of the stimulation keeping the frequency low and record a nice intensity-response function.
(3) Keeping the amplitude high enough, change the frequency and see if you can find tetanus (Fig 12-17 in your text)

Phan and Nicole got this series from Matt M in response to increasing stimulation at low frequency
Here is the result from that from Joel and his lab partners for tetanus using the force transducer

Writing comments on your record, saving your record, and making PhotoShop jpg's out of nice records was covered in the computer lab.

Last year, I gave a quiz and here are the answers.

BL A347 - General Physiology Laboratory Spring semester, 2004
Prof. Stark - second quiz - Feb 19
ANSWERS

From the back of the wrist to a place on the forearm, stimulation might cause one finger to move a little. In this lab, we expect to get small twitches if the current (mA) is low and bigger twitches if it is big. But in Fig. 12-17a&b twitches are the same size unless there is temporal summation. Why is our situation different from the book example?

The book example was axon to muscle fiber or nerve to muscle, but we can stimulate the muscle directly and recruit more fibers with higher stimulation

What is it called when a lot of twitches summate to achieve maximum tension?

tetanus

In the monosynaptic reflex as typified by the knee jerk, where is the motor neuron?

ventral horn of spinal cord

What ion flux is predominant in muscle acetylcholine receptors?

Na+

Some students in an animal physiology lab expose the frog gastrocnemius muscle and sciatic nerve, then they connect the nerve for stimulation. Also, they put electrodes in the muscle to stimulate muscle fibers directly. Everything works. After injecting curare, a nicotinic antagonist, into the muscle, what happens when the nerve is stimulated?...

nothing since ACh receptors are blocked

...and when the muscle is stimulated directly?

It works just fine since neuromuscular junction is bypassed

Suppose a subject for today's experiment has myasthenia gravis. What would happen to the response to nerve stimulation relative to a normal subject's responses?

It would be smaller since the lower # of ACh receptors would make neuromuscular junction work poorly

How would this be changed with neostigmine?

This ACh esterase inhibitor potentiates the ACh, in short supply in MG, so there is some recovery

Suppose you thought you were stimulating the muscle fibers directly, what would BoTox do to the response?

nothing since direct muscle stimulation makes the lack of vesicle release from BoTox irrelevant

How is Ca2+ important in the neuron terminal of the neuromuscular junction?

Ca2+ influx to terminal is part of the signal process leading to vesicle release


This page was last updated 9/8/05

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