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 and 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 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 has side effects like the impulse to gamble or take risks

Different Synapses for Different Purposes

Earlier 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 end-plate, 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.

We've also conducted our own research into this topic!

Dagda RK, Thalhauser RM, Dagda R, Marzullo TC, Gage GJ (2013) Using Crickets to Introduce Neurophysiology to Early Undergraduate Students. Journal of Undergraduate Neuroscience Education (JUNE), Fall 2013, 12(1):A66-A74.

Check out section 4 in this paper for more information about microstimulation!

Setup

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 as a means 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)

There are many programs that can generate electrical stimuli ideal for this lab. If you are using an iPhone or iPad, these free apps can be found at the iTunes store at the following links:

If you are using a PC, you can use this online software:

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:

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. Start with a song that has a lot of bass. Rap will work well - we like the Beastie Boys!
5. Next pick a song with a lot of treble. We found classical music works well for this!

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).
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.

Alternate 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.

Exercise 2: Response of Cockroach MNs 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 waves 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:

Below is an example with our results

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.