Microstimulation of Neurons and Muscles – Teacher Guide
Electricity plays a critical role how our nervous and muscle systems work. In this experiment you are
going to stimulate a cockroach's leg muscles using the music output of your mobile phone or computer.
Overview:
In this lab you will:
1. Learn how to prepare a cockroach leg for micro stimulation.
2. Test how different stimulations cause spikes in motor neurons.
3. Observe the difference between sensory and motor spiking patterns.
4. Investigate how different types of stimulations affect the generation of action potentials in a
cockroach leg preparation.
Objectives:
Before doing this lab you should understand:
 How neurons communicate with muscle cells.
 How electrical stimulation can cause a muscle contraction.
After doing this lab you should be able to:
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Explain how a nervous system controls muscle movement.
Describe how distinct movements are caused by specific spiking patterns.
Design an experiment to map ideal frequencies for stimulation.
Equipment:
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SpikerBox
Laptop Cable
Stimulation Cable
External speaker
Computer with Audacity
Installed
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Cockroaches
Ice water or Freezer
Dissection Scissors
Toothpick
Electric Signal Generator
(iPhone or MP3 players)
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External Speaker
(Optional)
Y-Splitter (Optional)
Additional Software:You will also need to install some type of software or app that allows you to
generate and alter signals. There are many types of here are many programs that can generate
electrical stimuli ideal for this lab.
For iPhone or iPad, these free apps can be found at the iTunes store:
 AudioSigGen
 FreqGen
If you are using a PC, you can use this online software:
 Rhintech
Additionally, you can simply download various frequencies as MP3s and play them through any MP3
player. Here is a website from which you can download free frequencies appropriate for this exercise:
 TestSound
Educational Standards
Please see our standards alignment guide at the back of this teacher guide for information on how this
lesson meets the Next Generation Science Standards and the Common Core Standards.
Introduction
Long before scientists were able to record spikes, they were able to stimulate the nervous system using
primitive batteries called Leyden Jars. Since nerves use electricity to communicate, they can be
manipulated with electricity as well. Luigi Galvani, an Italian scientist in the 1700's, discovered that
electricity applied to the nerves of frog legs caused the large muscles to twitch.
Such discoveries led to debates at the time as to whether "animal electricity" was different from the
electricity during lightning storms. Galvani decided to test this as well tested this by hanging frog legs
off his back porch during thunderstorms & watching the legs twitch. By demonstrating that the
electricity from lightening could also stimulate nerves, he showed that electricity was the same
whether it flowed through a nerve, or was produced in a thunderstorm.
Luigi Galvani’s Experimental Set Up:
Luigi Galvani tested whether electrical energy from storms was the same electricity found in nerves in
the body by running a wire up to the roof of his house and connecting it to nerves in frog legs. When
lightening struck the wire, the electricity stimulated the frog nerves causing the legs to move. His
experiments showed that the electricity found in nature and the electricity found in neural systems
are the same.
Galvani’s experiments were the inspiration for Mary Shelley's novel "Frankenstein”
"Perhaps a corpse would be re-animated; galvanism had given token of such
things: perhaps the component parts of a creature might be manufactured,
brought together, and endured with vital warmth."
-Mary Shelley, 1818
After demonstrating that electricity can indeed stimulate nervous system and muscle tissue, scientists
went on to prove that neural tissue generates its own electricity. This led to the beginnings of
contemporary neuroscience.
In another famous experiment, German medical scientists Eduard Hitzig and Gustav Fritsch in 1870
applied electric current to the exposed cerebral cortex (wrinkly part of brain) in dogs in their kitchens,
which people thought was odd even back then. Hitzig showed that stimulation of different parts of the
brain can cause different types of movements.
Today, such central-stimulation/motor-sensory-output techniques are used in patients with
neurological disease, most notably those afflicted with Parkinson's disease. By inserting a small, long
electrode into a specific part of the brain called the subthalamic nucleus, the shaking and tremors
associated with the disease can be lessened. However, sometimes stimulating the brain can also cause
side-effects, like increased gambling and other compulsive behaviors. Today, some advanced research
groups are designing small chips that stimulate the nerves of the eye as a cure for blindness.
Brain Stimulation in Medicine
Brain stimulation
has been used to
stimulate nerves that
are affected by
Parkinson’s disease,
but neural
stimulation also
have side effects like
the impulse to
gamble or take risks
Different Synapses for Different Purposes
During Lab One we learned about action
potentials (APs) and the neuron-neuron
synapses that exist in the central nervous
system and allow us to process stimuli.
However, if the neurons couldn’t also interact
with other types of cells, they would be useless.
The interface between neurons and muscle
tissue takes place in special synapses, called
neuromuscular junctions, that allow neurons to
stimulate muscle cells. In the central nervous
system, webs of interacting neurons (called
convergent connections) stimulate each other
and exchange information. However, a postsynaptic muscle cell is normally innervated (activated) by
just a single presynaptic motor neuron (MN).
Motor neurons connect to muscles at a specialized region of the muscle membrane called the endplate, and these specialized synapses between motor neurons and skeletal muscle cells are called
neuromuscular junctions. Acetylcholine (ACh) is released by the axon terminal from the MN, which
directly opens voltage-gated Ca+2 ion channels in the muscle membrane that allows Ca+2 to enter the
terminal with each action potential. Motor neurons excite the muscle by opening ion channels at the
end-plate, producing a large amplitude end-plate potential that rapidly activates voltage-gated Na+
channels and produces an action potential that propagates along the muscle fiber and generates
movement.
Directions
Microstimulation Electrode
The microstimulation electrode will act as a conduit between your electrical signal generator and the
cockroach. The electrode will consist of an audio cable that plugs into your signal generator to deliver
the stimulation to the cockroach. You can choose to solder either pins to your audio cable, similar to
those used in Lab 1, or use alligator clips that can be attached to the electrodes that come with your
SpikerBox.
Electrical Signal Generator (ESG)
Procedure
Exercise 1: Cockroach Leg Microstimulation Preparation
1. Prepare a cockroach leg and insert the electrodes as you did in Lab 1, getting started with the
SpikerBox.
2. Attach the micro stimulation electrode. If using the alligator clip electrode, attach the clips to
the SpikerBox recording electrodes inserted into the coxa and femur. If using a direct electrode,
place the electrodes into the coxa and femur of your cockroach leg.
3. Plug the Microstimulation Electrode into an ESG such as a computer or MP3 player. If you are
able to, program your ESG to produce square waves.
4. Begin by stimulating the cockroach leg with music
4a. First pick a song with a lot of bass (rap will work well –we like the Beastie Boys).
Name of Song:
No Sleep ‘Til Brooklyn
Artist:
Beastie Boys
Album:
Licensed to Ill
Cockroach Leg Movement:
Cockroach leg contracts and moves in time to the music.
4b. Now pick a song with a lot of treble (classical music is good for this – try Johan Sebastian
Bach).
Name of Song:
Aria BWV 998 - Goldberg Variations
Artist:
Johan Sebastian Bach
Album:
Goldberg Variations
Cockroach Leg Movement:
Leg moves only occasionally.
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Trouble-Shooting Your Cockroach Leg Set Up:
If your cockroach leg does not move in response to the audio stimulus, there are some simple checks
you can to ensure that your experimental system is set up correctly.
 Check Your Signal Generator: Check to be sure that you have your electrode plugged into the
audio out jack and that the tone is being produced. You should be able to hear a 200 Hz tone at half
volume with EXTERNAL SPEAKERS. If you do not hear a tone, you may need to adjust your audio
settings.
 Test Whether Your Cockroach Leg is Alive: If you can produce a tone with your ESG but the leg
still does not move then check to see that your cockroach leg is alive. Using the protocol in Lab 1:
Exercise 1, use your SpikerBox electrodes and speaker to ensure spikes are still being produced by
the neurons in the leg.
 Check your Electrode Placement: Check to be sure that you
have placed one electrode in the coxa and one in the femur (see
diagram to the right).
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Get Extra Help: Consult with your teacher if you continue to
have problems stimulating your cockroach leg.
After you have your cockroach leg apparatus working, please move on to exercise 2.
Note To Teachers: A Simplified Version of Exercise 1
You can run a simplified version of this experiment using a small speaker and asking the students to
whistle.
It may be helpful to explain how speakers work before beginning this activity. Sound is represented by
an electric current traveling through the wires. It then passes through a magnetic field in the speaker,
which causes the cone/drum to move, pushing the air and creating the sound that you hear. For
example, you can ask students if they have ever seen a bass woofer vibrate at a rock concert, or felt a
car shake when the bass was turned up to the maximum.
This principle also works in reverse, and this is how
microphones work. If you speak into a microphone/speaker,
the movement of the cone/drum inside causes a current to
flow in the wires.
Stimulating nerves this way requires a special speaker
called a piezoelectric, available at electronics stores. These
speakers can generate quite large voltages (1-3 V). Large
enough to actually excite nervous & muscle tissue!
For this simplified
experiment, connect the
two leads of the speaker to the needles in the cockroach leg using
alligator clip wire, place the speaker close to your mouth, and try to
whistle as loud as you can. Watch the leg; as you whistle louder and
louder, the leg should begin to move. Students may enjoy getting to
interact directly with the cockroach leg in this activity.
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Exercise 2: Response of Cockroach Motor Neurons to Amplitude and Frequency
To conduct real experiments, you’ll need more control over the stimulation than simply playing music.
Using the tone generation application you downloaded or the series of tones that you downloaded on
your phone or computer, you can carefully control the amount of stimulus that you give the cockroach
leg.
First, let’s cover how electrical waves vary before we begin our experiment. Waves can vary in several
ways, by frequency, or the width of the wave, or by height the amplitude of the wave. In this lab, we
will test which aspect of waves cockroach legs respond to. First, lets observe the wave’s we’ll
generating.
Put your settings on “square wave” and adjust the frequency or “width” of the wave
And you can also adjust the volume or amplitude (height)
Waves with the frequencies pictured below are used in deep brain stimulation to treat Parkinson’s
disease.
Using the tone generator, you will test the effect of frequency and volume on cockroach leg movement.
Begin stimulation of the leg at the lowest frequency (20 Hz) and volume (lowest amplitude your device
is capable of) shown in Table 1. Work your way through the rest of the combinations. Keep in mind, if
you were to try to listen to the output of your ESG, you may not be able to hear a tone of 20 Hz no
matter how loud you make it. Human hearing ranges between 20 Hz and 20,000 Hz (20 kHz), although
there is variation between individuals.
Use the table to keep track of your experiments. Add plusses where you see movement, and leave the
boxes blank where no movement occurs:
What is the best frequency and volume for stimulating the cockroach leg?
Frequency: Low frequencies (below 600) work best
Volume: At low frequencies, you can stimulate the leg at low volumes. At high frequency, more volume is
required to produce response.
Questions:
1. In this experiment you tested the responsiveness of the cockroach legs to a wide range of frequencies
and amplitudes. Was there any commonality in the types of frequencies and/or amplitudes that caused
the most responsiveness? Why do you think that might be?
Cockroach legs responded most strongly to low frequencies. In order to achieve movement at
higher frequencies, you had to increase the volume.
2. Were you able to identify any spiking patterns that led to motor movements? If so, what were the
characteristics of these patterns? What types of movements were you able to find these patterns for?
Spiking patterns that resulted in movements typically were high frequency (lots of little spikes
together). These were usually associated with leg contractions.
3. Some deaf people use a device called a cochlear implant to transmit sounds into electrical impulses
that their brain can process. In natural hearing, sound waves stimulate the ear drum, and then nerves in
the ear drum transmit sounds through the cochlear nerve to the brain. However, when a person’s ear
drum or surrounding nerves are damaged, they can no longer stimulate the cochlear nerve.
Acochlear implant is a small device that is surgically implanted in the ear of a person with hearing loss.
On the outside it has a sound processer that captures and records sound and then translates it into
electrical impulses, which are then transmitted to the cochlear nerve and on to the brain. The cochlear
implant bypasses damaged hearing cells in the ear drum and translates sounds directly into nerve
impulses that the brain can understand. Imagine you are a doctor and a patient comes to you
complaining that their cochlear implant is broken they can’t hear the high singing of their pet bird.
Would you adjust the frequency or the amplitude settings in their cochlear implant? What if they had
trouble hearing very loud sounds?
You would adjust the frequency settings. If they can’t hear the bird, which probably makes high frequency
sounds, then the frequency is probably set wrong. If they cannot hear loud sounds, the amplitude or
volume is likely the issue.