Synaptic Transmission Laboratory

Neurobiology 5004
Angie Ribera
Bill Betz
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Synaptic Transmission Laboratory

Lab handout
Much of our knowledge about synaptic structure and function has come from studies of the neuromuscular junction. In today's laboratory, we will impale living frog skeletal muscle fibers with a glass micropipette and record action potentials and synaptic potentials in four different bathing solutions: normal physiological saline (no additions), Mg/low calcium, curare, and neostigmine. For each solution, we will discuss relevant clinical situations that are mimicked by the additions. For example, curare, which blocks post-synaptic receptors and leads to flaccid muscle paralysis, is used on poison darts to paralyze prey by Yanomamo Indian hunters in South America, is used often in surgery for muscle relaxation, and also mimics the effects of a disease, myasthenia gravis, in which antibodies block receptors in a similar fashion to curare.
We will begin with a brief introduction to the lab, in which the preparation and equipment will be described, and the general procedures outlined. Then we will break into two groups; each group will work with one instructor on two setups.

I. Observations
A. Identify the components of the setup (micromanipulator, oscilloscope, stereo microscope, amplifiers, microelectrode, recording chamber with frog nerve-muscle preparation, instructor).

B. Looking through the microscope: Identify muscle fibers, nerve branches and myelinated axons, red blood cells, pigment cells (black), parasites (if any), tiny pins, nerve in Vaseline filled trough (why the Vaseline?). (use different magnifications as needed.)

C. Stimulate the nerve while looking through the microscope. Change the frequency of stimulation. At what frequency (approximately) do the single twitches fuse into a smooth tetanus?
NOTE: Instructions in square brackets refer to software and stimulator settings.
[Scope mode; adjust stimulus frequency slider; click stimulus on (button turns red) and off]

II. Impalements
Your instructor will demonstrate how to impale single muscle fibers and how to use the software. [Scope mode; turn Position knob on amplifier box to zero the voltage before impaling]

A. Resting potential (mV)?

B. Miniature end plate potentials (mepp's) present? [A/C radiobutton on]

Approximate amplitude (mV)?

Is this close to threshold for an AP?

C. Action Potential (AP): Stimulate the nerve once.
[Set DispTime to 0.1 second (click 0.1 radiobutton); depress the single toggle on the stimulator to get one shock]

Does the AP overshoot zero mV?

Look for a notch or inflection of the AP rising phase. What does it reflect?

Did the electrode come out of the cell (resting potential to ~0 mV)? If so, why?

Repeat part II a few times.

III. Unknown solutions
One preparation will receive the low calcium solution; the other will receive the curare solution. Before applying the solution, discuss the composition of each (i.e., the approximate concentrations of sodium, potassium, and calcium ions, and major anions, and curare).
Discuss experimental observations that would permit you to distinguish the two solutions.

Now do the experiment:
Impale a muscle fiber and look for mepps. Then change the bathing solution (your instructor will show you how to use the perfusion system). Observe the effects of the unknown solution on mepps:

Also observe the muscle through the microscope and stimulate the nerve with a single shock (depress the single toggle on the stimulator) - any contractions?

Which solution is it?

How can you rule out the other possibility?

IV. Wait for paralysis
The Mg/low calcium and curare solutions require 5-10 minutes to block synaptic transmission. Remove the electrode from the muscle fiber, and monitor the developing paralysis by stimulating the nerve every minute or so and watching the twitch through the microscope. When the twitching stops, the muscle is paralyzed.

The two solutions (Mg/low calcium and curare) are discussed sequentially below.

V. Mg/Low calcium preparation
Impale a muscle fiber.
A. Are there mepps? If so, are they normal amplitude?
[Scope mode; Click A/C radiobutton]

B. Stimulate the nerve at a frequency of about 1/sec and observe the end plate potentials (epps). Note amplitudes and rise times.
[EPP mode; lift repeat toggle on stimulator; click Hold On/off button to store and display repeated epps; click Erase Hold button to erase]

Compare the epp's with the mepp's. Can you observe discreet "steps" in the amplitudes of successive epp's? Are the "steps" about the size of a mepp?

C. Quantitative analysis: Collect a series of mepps [MEPP mode; adjust Threshold for detecting peaks of mepps; click Keepit button twice to keep results]

Collect a series of epps
[EPP mode; stimulate repeatedly at about 1 shock per second; adjust window (vertical red lines) so the epp peaks fall inside; click Keepit button once to keep peak values.]

Display the results of acquiring mepps and epps [Analyze mode]

D. Clinical discussion

{1. Hypocalcemia - IS OPTIONAL

a. Signs/symptoms

b. Mechanism

c. Causes

d. Rx
e. Does the Mg/Low Ca solution mimic hypocalcemia? If not, why? } - end optional part

2. Botulism

a. Signs/symptoms

b. Mechanism

c. Causes

d. Rx

e. Why is botulism relatively common in Colorado, compared to other places?

3. Myasthenic syndrome

a. Signs/symptoms

b. Mechanism

c. Causes

d. Rx

e. Facilitation: Stimulate the Mg/Low Ca preparation at high frequency.
[Scope mode; adjust stimulus frequency slider]

What happens to the epp amplitudes? Does the muscle contract? What is the cellular mechanism of this facilitation?

VI. Curare preparation
Impale a muscle fiber.
A. Are there mepp's?
[Scope mode; click A/C radiobutton]

B. Stimulate the nerve and observe epps. How do they compare with epp's in the Mg/Low Ca solution?
[EPP mode]

C. Two shock facilitation.
Stimulate about once a second, with different intervals between the dual shocks
[EPP mode; click Hold On; On the stimulator, move the Twin pulse knob down; change the Delay settings on the stimulator]

How long does facilitation last? Mechanism of facilitation?

Repeat the above a few times with different impalements.

D. Clinical discussion: Myasthenia gravis

1. Signs/symptoms

2. Mechanism

3. Cause

4. Rx

E. High frequency stimulation: Record control epps before a high frequency train, and follow the recovery from depression, which may overshoot to produce post-tetanic potentiation.

Deliver a train of stimuli at high frequency. Record a few epps before the train at low frequency (1/10 seconds) and after the train. Then click the Train button to deliver a single train of shocks at 30-50 per second.
[Scope mode; adjust stimulus frequency slider to 0.1 (click radiobutton); select Train parameters (frequency: 30-50 per second; duration 40-60 seconds)]

What happens to epp amplitudes during a high frequency train of stimuli?

Mechanism of synaptic depression?

Does the amplitude recover when the high frequency stimulation ends?

Mechanism of PTP?

Significance of PTP?

F. Anti-cholinesterase (Neostigmine).

1. What does acetylcholine esterase (AChE) normally do?

Where is AChE located at the synapse?

What do you predict will be the effects of blocking AChE?

What would be the effect of a therapeutic dose of Neostigmine on a normal person?

2. Impale a muscle fiber in the curare solution and observe epp amplitudes. Then perfuse the chamber with the same solution to which some Neostigmine has been added. What is the effect?
[EPP mode; Hold On]

3. Clinical effects of Neostigmine use.

a. Overdose: What effect of overdose do you predict? Add an excess of Neostigmine to the bath (your instructor will demonstrate how). What happens to epp amplitudes? What is the mechanism of the effect?

{b. Side effects of a therapeutic dose of Neostigmine. - IS OPTIONAL

There are two major types of ACh receptors: Nicotinic and Muscarinic

Where found in body?

Activated by ACh and?

Blocked by?

Effect of blocking AChE

c. What are the side effects of Neostigmine therapy in myasthenia gravis?

Now what do you predict would be the effect of a therapeutic dose of Neostigmine on a normal person?

Neostigmine doesn't cross the blood brain barrier. What would happen if it did?

4. Environmental hazards of anti-cholinesterases.

a. Organophosphate insecticides. Signs of poisoning?

What does SLUD or SLUDGE mean?

b. Nerve gas. Signs after inhalation of nerve gas?
Effect of making the nerve gas lipid soluble?} - end optional part